Human antibodies as diagnostic reagents

ABSTRACT

The invention provides methods of diagnosis using human antibodies. The methods are particularly useful for analyzing human samples containing HAMA or heterophilic antibodies. A human antibody can bind to an analyte in such samples without binding to HAMA or heterophilic antibodies present in the sample. The methods can be effected using a sandwhich format among others.

BACKGROUND

Although it has long been recognized that human antibodies are superiorto mouse antibodies for therapeutic use, the reverse has been thought tobe the case for in vitro diagnostics. The different perceived roles ofhuman and mouse antibodies reflect differences in their properties andmethods of preparation. The principal hitherto recognized advantages ofhuman antibodies relative to mouse antibodies are lack of humananti-mouse (HAMA) response on administration to a patient, longer invivo half-life, and better interaction with human complement. All ofthese advantages account for the superiority of human antibodies tomouse antibodies for therapeutic use, but none is relevant to use ofantibodies as reagents for in vitro diagnostics.

One of the principal advantages of mouse antibodies relative to humanantibodies is ease of isolation. Despite improvements in methods forproducing human antibodies in recent years, it has still generally beenconsidered to have been a simpler matter to produce a mouse antibodythan a human antibody, particularly when the desired antibody issparsely represented in the total repertoire of antibodies that must bescreened. Another advantage of mouse antibodies is that the mouseantibody constant region can be detected using a labelled anti-mouseantibody, typically prepared from another species, such as a goat, as adetection moiety. Such an antibody binds specifically to a mouseantibodies without binding to human antibodies present in the sample.Use of a secondary labelling moiety provides a useful format fordetecting analytes in a human tissue sample. A comparable format cannotbe used for human antibodies because an antibody against a humanconstant region would generate false positives by reacting with humanantibodies in the sample. Because of their simplicity of isolation andcompatibility with detection using a secondary labelling moiety, andbecause properties such as generation of a HAMA response, in vivo halflife and complement activation are irrelevant for in vitro diagnosticpurposes, mouse antibodies have been used for in vitro diagnostics tothe virtual or total exclusion of human antibodies.

Although mouse antibodies are now in widespread use as diagnosticreagents, some problems have arisen when such antibodies are used todetect an analyte in a human sample. In some human samples, falsepositive or negative results are obtained due to the presence of HAMA orheterophilic antibodies in the sample. HAMA antibodies may be present ina human sample due to prior treatment of the patient from whom thesample was obtained with a mouse antibody (unrelated to the mouseantibody being used in diagnosis) or by environmental exposure to mouseantigens. Heterophilic antibodies are present in some patients as aresponse to certain pathogenic infections, such as Epstein Barr virus.Either HAMA or heterophilic antibodies in a sample can bind to a mouseantibody being used as a diagnostic reagent thereby generating a falsepositive signal. In sandwich assay formats, HAMA or heterophilicantibodies can form a bridge between immoblized and solution antibodiesto generate a false positive, as in other formats. Alternatively, in asandwich assay format, some HAMA or heterophilic antibodies may bind tothe immobilized antibody without binding to the solution antibody (orvice versa) thereby preventing immobilized antibody and solutionantibody from bridging to each other through an analyte and thusgenerating a false negative. In consequence, a significant number ofassays performed on human clinical samples using mouse antibodies as thediagnostic reagent generate inaccurate results.

U.S. patent application Ser. No. 09/453,234, filed Dec. 1, 1999, U.S.Ser. No. 60/157,415, filed Oct. 2, 1999, PCT 98/06704, filed, Apr. 3,1998, U.S. Ser. No. 08/835,159, filed Apr. 4, 1997 (now abandoned) andU.S. Ser. No. 08/832,985, filed Apr. 4, 1997 (now U.S. Pat. No.6,057,098) are directed to related subject matter, and each isincorporated by reference in its entirety for all purposes.

SUMMARY OF THE INVENTION

The invention provides methods of detecting an analyte in a human samplecontaining human antibodies that specifically bind to antibodies from anonhuman species. Such methods entail contacting the sample with a humanantibody. The human antibody specifically binds to the analyte withoutspecifically binding to the human antibodies that specifically bind toantibodies from a nonhuman species (e.g., HAMA or heterophilicantibodies present in the sample). Binding between the human antibodyand the analyte is then detected. In some methods, the sample iscontacted with a first human antibody that is immobilized on a supportand a second human antibody in solution wherein the first and secondhuman antibodies bind to different epitopes on the analyte; and thedetecting step detects binding between the first and/or second humanantibody to the analyte. In such methods, the second antibody istypically labelled. In some methods, the sample is contacted with afirst population of human antibodies immobilized to a support and asecond population of human antibodies in solution, wherein members fromthe first and second populations bind to different epitopes on theanalyte.

Human antibodies used in such methods typically have affinities of atleast 10⁸ M⁻¹, 10⁹ M⁻¹, 10¹⁰ M⁻¹, 10¹¹M⁻¹, 10¹² M⁻¹ for the analyte.Some human antibodies used in the methods are produced by expression ofa recombinant construct in E. coli. Some such antibodies haveimmunoreactivities of at least 90%.

The invention further provides methods of detecting an analyte in asample Such methods entail contacting the sample with a first humanantibody immobilized to a solid phase, and a second human antibody insolution, wherein the first and second antibodies bind to differentepitopes of the analyte if present in the sample. Binding of the analyteto the first and/or second antibodies is then detected. Bindingindicates presence of the analyte in the sample. In such methods, thesecond antibody is typically labelled and the detecting detects bindingof second antibody to the analyte. In some methods, the sample iscontacted with a first population of human antibodies immobilized to asupport and a second population of human antibodies in solution, whereinmembers from the first and second populations bind to different epitopeson the analyte in some methods, the binding of the first and/or secondhuman antibodies to the analyte reaches equilibrium within an hour.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: shows a vector obtained from Ixsys, Inc. and described in Huse,WO 92/06204, which provides the starting material for producing phagedisplay vectors. The following abbreviations are used:

A. Nonessential DNA sequence later deleted.

B. Lac promoter and ribosome binding site.

C. Pectate lyase signal sequence.

D. Kappa chain variable region.

E. Kappa chain constant region.

F. DNA sequence separating kappa and heavy chain, includes ribosomebinding site for heavy chain.

G. Alkaline phosphatase signal sequence.

H. Heavy chain variable region.

I. Heavy chain constant region including 5 amino acids of the hingeregion.

J. Decapeptide DNA sequence.

K. Pseudo gene VIII sequence with amber stop codon at 5′ end.

L. Nonessential DNA sequence that was later deleted.

FIG. 2: Oligonucleotides used in vector construction.

FIG. 3: Map of the vector pBRncoH3.

FIG. 4: Insertion of araC into pBR-based vector (FIG. 4A) and theresulting vector pBRnco (FIG. 4B).

FIG. 5: Subcloning of a DNA segment encoding a Fab by T4 exonucleasedigestion.

FIG. 6 Targeted insertion of a neo cassette into the SmaI site of themu1 exon. A. Schematic diagram of the genomic structure of the mu locus.The filled boxes represent the mu exons. B. Schematic diagram of theCmuD targeting vector. The dotted lines denote those genomic musequences included in the construct. Plasmid sequences are not shown. C.Schematic diagram of the targeted mu locus in which the neo cassette hasbeen inserted into mu1. The box at the right shows those RFLP'sdiagnostic of homologous recombination between the targeting constructand the mus locus. The FGLP's were detected by Southern blothybridization using probe A, the 915 SaI fragment shown in C.

FIG. 7 Nongermline encoded nucleotides in heavy and light chain V genes.Heavy chain V genes were found to be heavily somatically mutated. Lightchain V genes comprised fewer non-germline encoded nucleotides.

FIG. 8: Calibration curve for different concentrations of IL-8 detectedusing polyclonal mouse antibodies in sandwich assay.

FIG. 9: Calibration curve for different concentrations of IL-8 detectedusing polyclonal human antibodies in sandwhich assay.

FIG. 10: Calibration curve for different concentrations of IL-8 detectedusing two monoclonal human antibodies in a sandwich assay.

DEFINITIONS

Specific binding between an antibody or other binding agent and anantigen means a binding affinity of at least 10⁶ M⁻¹. Preferred bindingagents bind with affinities of at least about 10⁷ M⁻¹, and preferably10⁸ M⁻¹ to 10⁹ M⁻¹, 10¹⁰ M⁻¹, 10¹¹ M⁻¹, or 10¹² M⁻¹. The term epitopemeans an antigenic determinant capable of specific binding to anantibody. Epitopes usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three dimensional structural characteristics, aswell as specific charge characteristics. Conformational andnonconformational epitopes are distinguished in that the binding to theformer but not the latter is lost in the presence of denaturingsolvents.

The basic antibody structural unit is known to comprise a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 Kda). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxyl-terminal portion of each chain definesa constant region primarily responsible for effector function.

Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, and define theantibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 10 more amino acids. (See generally,Fundamental Immunology (Paul, W., ed., 2nd ed. Raven Press, N.Y., 1989,4th edition (1999), Paul William E., ed. Raven Press, N.Y.,(incorporated by reference in its entirety for all purposes). The genesencoding variable regions of heavy and light immunoglobulin chains arereferred to as V_(H) and V_(L) respectively. Although the amino acidsequence of an immunoglobulin chain is not exactly the same as would bepredicted from the V_(H) or V_(L) gene that encoded it due to somaticmutations (see FIG. 7), there is sufficient similarity between predictedand actual sequences of immunoglobulins that the actual sequence ischaracteristic and allows recognition of a corresponding V_(H) or V_(L)gene. The term constant region is used to refer to both full-lengthnatural constant regions and segments thereof, such as C_(H)1, hinge,C_(H)2 and C_(H)3 or fragments thereof. Typically, segments of light andheavy chain constant regions in antibodies have sufficient length tocontribute to interchain bonding between heavy and light chain.

The variable regions of each light/heavy chain pair form the antibodybinding site. Thus, an intact antibody has two binding sites. Except inbifunctional or bispecific antibodies, the two binding sites are thesame. The chains all exhibit the same general structure of fourrelatively conserved framework regions (FR) joined by threehypervariable regions, also called complementarily determining regionsor CDRs. The CDRs from the two chains of each pair are aligned by theframework regions, enabling binding to a specific epitope. CDR and FRresidues are delineated according to the standard sequence definition ofKabat, et al., supra. An alternative structural definition has beenproposed by Chothia, et al., J. Mol. Biol. 196:901-917 (1987); Nature342:878-883 (1989); and J. Mol. Biol. 186:651-663 (1989).

The term antibody is used to mean whole antibodies and binding fragmentsthereof. Binding fragments include single chain fragments, Fv fragmentsand Fab fragments. The term Fab fragment is sometimes used in the art tomean the binding fragment resulting from papain cleavage of an intactantibody. The terms Fab′ and F(ab′)2 are sometimes used in the art torefer to binding fragments of intact antibodies generated by pepsincleavage. Here, Fab is used to refer generically to double chain bindingfragments of intact antibodies having at least substantially completelight and heavy chain variable domains sufficient for antigen-specificbindings, and parts of the light and heavy chain constant regionssufficient to maintain association of the light and heavy chains.Usually, Fab fragments are formed by complexing a full-length orsubstantially full-length light chain with a heavy chain comprising thevariable domain and at least the C_(H)1 domain of the constant region.

An isolated species or population of species means an object species(e.g., binding polypeptides of the invention) that is the predominantspecies present (i.e., on a molar basis it is more abundant than otherspecies in the composition). Preferably, an isolated species comprisesat least about 50, 80 or 90 percent (on a molar basis) of allmacromolecular species present. Most preferably, the object species ispurified to essential homogeneity (contaminant species cannot bedetected in the composition by conventional detection methods). A targetis any molecule for which it is desired to isolate partners withspecific binding affinity for the target.

Targets of interest include antibodies, including anti-idiotypicantibodies and autoantibodies present in autoimmune diseases, such asdiabetes, multiple sclerosis and rheumatoid arthritis. Other targets ofinterest are growth factor receptors (e.g., FGFR, PDGFR, EFG, NGFR, andVEGF) and their ligands. Other targets are G-protein receptors andinclude substance K receptor, the angiotensin receptor, the α- andβ-adrenergic receptors, the serotonin receptors, and PAF receptor. See,e.g., Gilman, Ann. Rev. Biochem. 56:625-649 (1987). Other targetsinclude ion channels (e.g., calcium, sodium, potassium channels),muscarinic receptors, acetylcholine receptors, GABA receptors, glutamatereceptors, and dopamine receptors (see Harpold, U.S. Pat. No. 5,401,629and U.S. Pat. No. 5,436,128). Other targets are adhesion proteins suchas integrins, selecting, and immunoglobulin superfamily members (seeSpringer, Nature 346:425-433 (1990). Osborn, Cell 62:3 (1990); Hynes,Cell 69:11 (1992)). Other targets are cytokines, such as interleukinsIL-1 through IL-13, tumor necrosis factors α & β, interferons α, β andγ, tumor growth factor Beta (TGF-β), colony stimulating factor (CSF) andgranulocyte monocyte colony stimulating factor (GM-CSF). See HumanCytokines: Handbook for Basic & Clinical Research (Aggrawal et al. eds.,Blackwell Scientific, Boston, Mass. 1991). Other targets are hormones,enzymes, and intracellular and intercellular messengers, such as, adenylcyclase, guanyl cyclase, and phospholipase C. Drugs are also targets ofinterest. Target molecules can be human, mammalian or bacterial. Othertargets are antigens, such as proteins, glycoproteins and carbohydratesfrom microbial pathogens, both viral and bacterial, and tumors. Stillother targets are described in U.S. Pat. No. 4,366,241. Some agentsscreened by the target merely bind to a target. Other agents agonize orantagonize the target.

Display library members having full-length polypeptide coding sequenceshave coding sequences the same length as that of the coding sequencesoriginally inserted into a display vector before propagation of thevector.

The term phage is used to refer to both phage containing infectivegenomes and phage containing defective genomes that can be packaged onlywith a helper phage. Such phage are sometimes referred to as phagemids.

The term “human antibody” includes antibodies having variable andconstant regions (if present) derived from human germline immunoglobulinsequences. Human antibodies of the invention can include amino acidresidues not encoded by human germline immunoglobulin sequences (e.g.,mutations introduced by random or site-specific mutagenesis in vitro orby somatic mutation in vivo). However, the term “human antibody” doesnot include antibodies in which CDR sequences derived from the germlineof another mammalian species, such has a mouse, have been grafted ontohuman framework sequences (i.e., humanized antibodies).

A rearranged heavy chain or light chain immunoglobulin locus has a Vsegment positioned immediately adjacent to a D-J or J segment in aconformation encoding essentially a complete V_(H) or V_(L) domain,respectively. A rearranged immunoglobulin gene locus can be identifiedby comparison to germline DNA; the rearranged locus having at least onerecombined heptamer/nonamer homology element. Conversely, anunrearranged or germline configuration refers to a configuration inwhich the V segment is not recombined so as to be immediately adjacentto a D or J segment.

“Isotype switching” refers to the phenomenon by which the class, orisotype, of an antibody changes from one Ig class to one of the other Igclasses.

“Nonswitched isotype” refers to the isotypic class of heavy chain thatis produced when no isotype switching has taken place; the C_(H) geneencoding the nonswitched isotype is typically the first CH geneimmediately downstream from the functionally rearranged VDJ gene.Isotype switching has been classified as classical or non-classicalisotype switching. Classical isotype switching occurs by recombinationevents which involve at least one switch sequence region in thetransgene. Non-classical isotype switching may occur by, for example,homologous recombination between human σ_(μ) and human Σ_(μ)(δ-associated deletion). Alternative non-classical switching mechanisms,such as intertransgene and/or interchromosomal recombination, amongothers, may occur and effectuate isotype switching.

The term “switch sequence” refers to those DNA sequences responsible forswitch recombination. A “switch donor” sequence, typically a μ switchregion, are 5′ (i.e., upstream) of the construct region to be deletedduring the switch recombination. The “switch acceptor” region arebetween the construct region to be deleted and the replacement constantregion (e.g., γ, ε, etc.). As there is no specific site whererecombination always occurs, the final gene sequence is not typicallypredictable from the construct.

Competition is determined by an assay in which the antibody under testinhibits specific binding of a reference antibody to a given target.Numerous types of competitive binding assays are known for example: (seeHarlow and Lane, Antibodies, A Laboratory Manual, Cold Spring HarborPress (1988)). Typically, such an assay involves the use of purifiedtarget, an unlabelled test antibody and a labeled reference antibody.Competitive inhibition is measured by determining the amount of labelbound to targetin the presence of the test antibody. Usually the testantibody is present in excess. Antibodies identified by competitionassay (competing antibodies) include antibodies binding to the sameepitope as a reference antibody and antibodies binding to an adjacentepitope sufficiently proximal to the epitope bound by the referenceantibody for steric hindrance to occur. When a competing antibody ispresent in excess, it will inhibit specific binding of a referenceantibody to the target by 50%.

DETAILED DESCRIPTION I. General

The present invention provides methods of diagnosis using humanantibodies as the diagnostic reagent(s). The methods are premised, inpart, on the insight that use of human antibodies avoids substantialdistortions in measured analyte concentrations due to HAMA and/orheterophilic antibodies present in some human samples. Data showing theextent of such distortions and how they are overcome by the use of humanantibodies are provided in Example 28.

Inefficiencies and limitations of prior methods of generating humanantibodies relative to mouse antibodies are overcome by the disclosureof new methods for producing indefinite numbers of human antibodieshaving extraordinarily high binding affinities and multiple epitopespecificities. Examples of the affinities of human antibodies producedby these methods are described in Example 21. Evidence that theantibodies bind to a number of different epitopes is provided by Example27. The use of high affinities antibodies as diagnotic reagents isdesirable because it allows washing to be conducted at higher stringencyand consequently results in a higher signal to noise ratio. Use ofantibodies binding to different epitopes is advantageous sandwhichdetection formats. Using the methods disclosed in U.S. Ser. No.60/157415, filed Oct. 2, 1999 and reproduced in the present application,high affinity human antibodies, optionally binding to differentepitopes, can be generated with comparable facility to that of mouseantibodies generated by Milstein-Kohler technology.

A further advantage of human antibodies for diagnostic assays disclosedby the present application is the result that human antibodies producedby recombinant expression in bacteria, such as E. coli, fold to produceantibody specifically immunoreactive with antigen at high efficiency,greater than that which is typical for mouse antibodies. Examples of theextent of immunoreactivity of human antibodies expressed in E. coli areprovided in Table 4. Use of antibodies with high immunoreactive isadvantageous in increasing the signal to noise ratio in a diagnosticassay.

II. Production of Human Antibodies

A. General

Methods for producing human antibodies are described in copendingapplication U.S. Ser. No. 60/157415, filed Oct. 2, 1999. These methodsinclude the trioma technology of Ostberg et al. (1983), Hybridoma2:361-367 and Engelman et al., U.S. Pat. No. 4,634,666, phage displaymethods and nonhuman transgenic mice expressing genes of the humanimmune system. Preferred methods are reproduced below. The methodstypically work by immunizing a nonhuman transgenic animal having humanimmunoglobulin genes. The animal expresses a diverse range of humanantibodies that bind to the antigen. Nucleic acids encoding the antibodychain components of such antibodies are then cloned from the animal intoa display vector. Typically, separate populations of nucleic acidsencoding heavy and light chain sequences are cloned, and the separatepopulations then recombined on insertion into the vector, such that anygiven copy of the vector receives a random combination of a heavy andlight chains. The vector is designed to express antibody chains so thatthey can be assembled and displayed on the outersurface of a displaypackage containing the vector. For example, antibody chains can beexpressed as fusion proteins with a phage coat protein from theoutersurface of the phage. Thereafter, display packages can be screenedfor display of antibodies binding to a target.

In some methods, display packages are subject to a prescreening step. Insuch methods, the display package encode a tag expressed as a fusionprotein with an antibody chain displayed from the package. Displaypackages are prescreened for binding to a receptor to the tag. It isbelieved that the prescreening serves to enrich for display packagesdisplaying multiple copies of an antibody chain linked to the tag, andthat it is this subset of display packages that binds to target in thesubsequent screening step. However, practice of the invention is notdependent on whether this mechanism is correct.

After prescreening with receptor (if any) and screening with target,display packages binding to the target are isolated, and optionally,subject to further rounds screening to target, with each such roundoptionally being preceded by prescreening to receptor. Display packagesare typically amplified between rounds of screening to target but notbetween prescreening and screening steps. After one or a few rounds ofscreening to target, the remaining display packages are highly enrichedfor high affinity binders to the target. For example, as shown inExample 13, it is possible to isolate large numbers of differentantibodies having affinities in excess of 10⁹ or 10¹⁰ M⁻¹. Furthermore,the conditions of screening can be controlled to select antibodieshaving affinity in excess of a chosen threshold.

In some methods, nucleic acids encoding human antibody chains aresubcloned en masse from display vectors surviving selection to anexpression vector. Typically, a nucleic acid encoding both heavy andlight chains of an antibody displayed from a display package issubcloned to an expression vector thereby preserving the samecombinations of heavy and light chains in expression vectors as werepresent in the display packages surviving selection. The expressionvector can be designed to express inserted antibody chains as Fabfragments, intact antibodies or other fragments. Cloning en masse ofnucleic acids encoding antibody chains into an expression vector andsubsequent expression of the vector in host cells results in apolyclonal population of intact human antibodies or fragments thereof.Such a population contains a diverse mixture of different antibodytypes, the majority of which types show very high affinity for the sametarget, albeit usually to different epitopes within the target.

It is believed that the success of the invention in providing virtuallyunlimited numbers of unusually high affinity human antibodies to anydesired target (see Example 21) results, in part, from the combinationof display and transgenic animal approaches. Display methods provide ameans for screening vast numbers of antibodies for desired properties.However, the random association of light and heavy chains that occurs oncloning into a display vector results in unnatural combinations of heavyand light chains that may be nonfunctional. When heavy and light chainsare cloned from a natural human, the number of permutations of heavy andlight chains is very high, and probably a very large proportion of theseare nonnaturally occurring and not capable of high affinity binding.Thus, high affinity antibodies constitute a very small proportion ofsuch libraries and are difficult to isolate. Nonhuman transgenic animalswith human immunoglobulin genes typically do not include the fullcomplement of human immunoglobulin genes present in a natural human. Itis believed that the more limited complement of human immunoglobulingenes present in such animals results in a reduced proportion ofunnatural random permutations of heavy and light chains incapable ofhigh affinity binding. Thus, when the vast power of display selection isapplied free of the burden of very large numbers of unnaturalcombinations inherent in previous methods, indefinitely large numbers ofhuman immunoglobulins having very high affinities result.

Somatic mutation and affinity maturation of antibody genes allows forthe evolutionary selection of variant sequences based on bindingaffinity. However, this process differs from evolutionary naturalselection of individuals from sexually reproducing species because thereis no mechanism to allow for the combination of separately selectedbeneficial mutations. The absence of recombination between individual Bcells requires that beneficial mutations be selected for sequentially.Theoretically, combinatorial libraries allow for such combinations (atleast in the case where the two mutations are on heavy and light chainsrespectively). However, combinatorial libraries derived from naturalsources include such a wide diversity of different heavy/light chainpairs that the majority of the clones are not derived from the same Bcell bone marrow precursor cell. Such pairings are less likely to formstable antibody molecules that recognize the target antigen. Transgenicanimals that comprise B cell populations derived from a smaller numberof bone marrow precursors may be particularly useful for generatinglibraries that include novel, somatically mutated, heavy/light chainpairs in which each chain is derived from descendants of the sameoriginal pre-B cell.

Although the above mechanism is believed to explain the results achievedusing the invention, practice of the invention is not dependent on thecorrectness of this belief.

B. Transgenic Animals with Human Immune Systems

The transgenic animals used in the invention bear a heterologous humanimmune system and typically a knocked out endogenous immune systems.Mice are a preferred species of nonhuman animal. Such transgenic micesometimes referred to as HuMAb mice contain a human immunoglobulin geneminiloci that encodes unrearranged human heavy (μ and γ) and κ lightchain immunoglobulin sequences, together with targeted mutations thatinactivate the endogenous μ and κ chain loci (Lonberg et al. (1994)Nature 368(6474): 856-859 and U.S. Pat. No. 5,770,429). Accordingly, themice exhibit reduced expression of mouse IgM or κ, and in response toimmunization, the introduced human heavy and light chain transgenesundergo class switching and somatic mutation to generate high affinityhuman IgGκ monoclonal (Lonberg et al. (1994), supra; reviewed inLonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101;Lonberg and Huszar,. (1995) Intern. Rev. Immunol. Vol. 13: 65-93, andHarding and Lonberg (1995) Ann. N.Y. Acad. Sci 764:536-546); Taylor, L.et al. (1992) Nucleic Acids Research 20:6287-6295; Chen, J. et al.(1993) International Immunology 5: 647-656; Tuaillon et al. (1993) Proc.Natl. Acad. Sci USA 90:3720-3724; Choi et al. (1993) Nature Genetics4:117-123; Chen, J. et al. (1993) EMBO J. 12: 821-830; Tuaillon et al.(1994) J. Immunol. 152:2912-2920; Lonberg et al., (1994) Nature368(6474): 856-859; Lonberg, N. (1994) Handbook of ExperimentalPharmacology 113:49-101; Taylor, L. et al. (1994) InternationalImmunology 6: 579-591; Lonberg, N. and Huszar, D. (1995) Intern. Rev.Immunol. Vol. 13: 65-93; Harding, F. and Lonberg, N. (1995) Ann. N.Y.Acad. Sci 764:536-546; Fishwild, D. et al. (1996) Nature Biotechnology14:845-851; U.S. Pat. Nos. 5,625,126 and 5,770,429 U.S. Pat. No.5,545,807, U.S. Pat. No. 5,939,598, WO 98/24884, WO 94/25585, WO93/1227, WO 92/22645, WO 92/03918, the disclosures of all of which arehereby incorporated by reference in their entity.

Some transgenic non-human animals are capable of producing multipleisotypes of human monoclonal antibodies to an antigen (e.g., IgG, IgAand/or IgE) by undergoing V-D-J recombination and isotype switching.Isotype switching may occur by, e.g., classical or non-classical isotypeswitching.

Transgenic non-human animal are designed so that human immunoglobulintransgenes contained within the transgenic animal function correctlythroughout the pathway of B-cell development. In some mice, correctfunction of a heterologous heavy chain transgene includes isotypeswitching. Accordingly, the transgenes of the invention are constructedso as to produce isotype switching and one or more of the following: (1)high level and cell-type specific expression, (2) functional generearrangement, (3) activation of and response to allelic exclusion, (4)expression of a sufficient primary repertoire, (5) signal transduction,(6) somatic hypermutation, and (7) domination of the transgene antibodylocus during the immune response.

In transgenic animals in which the endogenous immunoglobulin loci of thetransgenic animals are functionally disrupted, the transgene need notactivate allelic exclusion. Further, in transgenic animals in which thetransgene comprises a functionally rearranged heavy and/or light chainimmunoglobulin gene, the second criteria of functional generearrangement is unnecessary, at least for transgenes that are alreadyrearranged.

Some transgenic non-human animals used to generate the human monoclonalantibodies contain rearranged, unrearranged or a combination ofrearranged and unrearranged heterologous immunoglobulin heavy and lightchain transgenes in the germline of the transgenic animal. In addition,the heavy chain transgene can contain functional isotype switchsequences, which are capable of supporting isotype switching of aheterologous transgene encoding multiple CH genes in the B-cells of thetransgenic animal. Such switch sequences can be those which occurnaturally in the germline immunoglobulin locus from the species thatserves as the source of the transgene CH genes, or such switch sequencescan be derived from those which occur in the species that is to receivethe transgene construct (the transgenic animal). For example, a humantransgene construct that is used to produce a transgenic mouse mayproduce a higher frequency of isotype switching events if itincorporates switch sequences similar to those that occur naturally inthe mouse heavy chain locus, as presumably the mouse switch sequencesare optimized to function with the mouse switch recombinase enzymesystem, whereas the human switch sequences are not. Switch sequences canbe isolated and cloned by conventional cloning methods, or can besynthesized de novo from overlapping synthetic oligonucleotides designedon the basis of published sequence information relating toimmunoglobulin switch region sequences (Mills et al., Nucl. Acids Res.15:7305-7316 (1991); Sideras et al., Intl. Immunol. 1:631-642 (1989)incorporated by reference). Typically, functionally rearrangedheterologous heavy and light chain immunoglobulin transgenes are foundin a significant fraction of the B-cells of the above transgenic animal(at least 10 percent).

The transgenes used to generate the transgenic animals of the inventioninclude a heavy chain transgene comprising DNA encoding at least onevariable gene segment, one diversity gene segment, one joining genesegment and at least one constant region gene segment. Theimmunoglobulin light chain transgene comprises DNA encoding at least onevariable gene segment, one joining gene segment and at least oneconstant region gene segment. The gene segments encoding the light andheavy chain gene segments are heterologous to the transgenic non-humananimal in that they are derived from, or correspond to, DNA encodingimmunoglobulin heavy and light chain gene segments from a species otherthan the transgenic non-human animal, typically the human species.

Typically transgenes are constructed so that the individual genesegments are unrearranged, i.e., not rearranged so as to encode afunctional immunoglobulin light or heavy chain. Such unrearrangedtransgenes support recombination of the V, D, and J gene segments(functional rearrangement) and preferably support incorporation of allor a portion of a D region gene segment in the resultant rearrangedimmunoglobulin heavy chain within the transgenic non-human animal whenexposed to antigen. Such transgenes typically comprise a substantialportion of the C, D, and J segments as well as a subset of the V genesegments.

In such transgene constructs, the various regulatory sequences, e.g.promoters, enhancers, class switch regions, splice-donor andsplice-acceptor sequences for RNA processing, recombination signals andthe like, comprise corresponding sequences derived from the heterologousDNA. Such regulatory sequences can be incorporated into the transgenefrom the same or a related species of the non-human animal used in theinvention. For example, human immunoglobulin gene segments can becombined in a transgene with a rodent immunoglobulin enhancer sequencefor use in a transgenic mouse. Alternatively, synthetic regulatorysequences can be incorporated into the transgene, wherein such syntheticregulatory sequences are not homologous to a functional DNA sequencethat is known to occur naturally in the genomes of mammals. Syntheticregulatory sequences are designed according to consensus rules, such as,for example, those specifying the permissible sequences of asplice-acceptor site or a promoter/enhancer motif. The transgene cancomprise a minilocus.

Some transgenic animals used to generate human antibodies contain atleast one, typically 2-10, and sometimes 25-50 or more copies of thetransgene described in Example 37 of U.S. Pat. No. 5,770,429, or thetransgene described in Example 24 (e.g., HCo12), at least one copy of alight chain transgene described in Examples 38 of U.S. Pat. No.5,770,429, two copies of the Cmu deletion described in Example 23, andtwo copies of the Jkappa deletion described in Example 9 of U.S. Pat.No. 5,770,429, each incorporated by reference in its entirety for allpurposes.

Some transgenic animals exhibit immunoglobulin production with asignificant repertoire. Thus, for example, animals in which theendogenous Ig genes Save been inactivated, the total immunoglobulinlevels range from about 0.1 to 10 mg/ml of serum, preferably 0.5 to 5mg/ml. The immunoglobulins expressed by the transgenic mice typicallyrecognize about one-half or more of highly antigenic proteins, e.g.,staphylococcus protein A.

The transgenic nonhuman animals can be immunized with a purified orenriched preparation of antigen and/or cells expressing antigen. Theanimals produce B cells that undergo class-switching via intratransgeneswitch recombination (cis-switching) and express immunoglobulinsreactive with the antigen with which they are immunized. Theimmunoglobulins can be human sequence antibodies, in which the heavy andlight chain polypeptides are encoded by human transgene sequences, whichcan include sequences derived by somatic mutation and V regionrecombinatorial joints, as well as germline-encoded sequences. Thesehuman sequence immunoglobulins can be referred to as being substantiallyidentical to a polypeptide sequence encoded by a human V_(L) or V_(H)gene segment and a human JL or JL segment, even though othernon-germline sequences may be present as a result of somatic mutationand differential V-J and V-D-J recombination joints. With respect tosuch human sequence antibodies, the variable regions of each chain aretypically at least 80 percent encoded by human germline V, J, and, inthe case of heavy chains, D, gene segments; frequently at least 85percent of the variable regions are encoded by human germline sequencespresent on the transgene; often 90 or 95 percent or more of the variableregion sequences are encoded by human germline sequences present on thetransgene. However, since non-germline sequences are introduced bysomatic mutation and VJ and VDJ joining, the human sequence antibodiesfrequently have some variable region sequences (and less frequentlyconstant region sequences) which are not encoded by human V, D, or Jgene segments as found in the human transgene(s) in the germline of themice. Typically, such non-germline sequences (or individual nucleotidepositions) cluster in or near CDRs, or in regions where somaticmutations are known to cluster.

The human sequence antibodies which bind to the predetermined antigencan result from isotype switching, such that human antibodies comprisinga human sequence γ chain (such as γ1, γ2, γ3, or γ4) and a humansequence light chain (such as kappa or lamda) are produced. Suchisotype-switched human sequence antibodies often contain one or moresomatic mutation(s), typically in the variable region and often in orwithin about 10 residues of a CDR) as a result of affinity maturationand selection of B cells by antigen, particularly subsequent tosecondary (or subsequent) antigen challenge. FIG. 7 shows the frequencyof somatic mutations in various immunoglobulins of the invention.

HuMAb transgenic animals can be immunized intraperitoneally (IP) withantigen in complete Freund's adjuvant, followed by IP immunizations withantigen in incomplete Freund's adjuvant every two weeks or month for afew months. Adjuvants other than Freund's are also effective. Inaddition, whole cells in the absence of adjuvant are found to be highlyimmunogenic. The immune response can be monitored over the course of theimmunization protocol with plasma samples being obtained by retroorbitalbleeds. Mice can be boosted intravenously with antigen 3 days beforesacrifice and removal of the spleen. 2-3 fusions for each immunizationare typically performed.

Nucleic acids encoding at least the variable regions of heavy and lightchains can be cloned from either immunized or naive transgenic animals.Nucleic acids can be cloned as genomic or cDNA from lymphatic cells ofsuch animals. The spleen is a preferred source of such cells. Noimmortalization of such cells is required prior to cloning ofimmunoglobulin sequences. Usually, mRNA is isolated and amplified byreverse transcription with polydT primers. The cDNA is then amplifiedusing primers to conserved regions of human immunoglobulins. Althoughpopulations of light and heavy chains can be amplified separately fromeach, the light chains within the light chain population are amplifieden masse as are the heavy chains within the heavy chain population.Typically, the amplified population of light chains comprises at least100, 1000, 10,000, 100,000 or 1,000,000 different light chains.Likewise, the amplified population of heavy chains comprises at least100, 1000, 10,000, 100,000 or 1,000,000 different heavy chains.

C. Display Libraries 1. Display Packages

A display package, sometimes referred to as a replicable geneticpackage, is a screenable unit comprising a polypeptide to be screenedlinked to a nucleic acid encoding the polypeptide. The nucleic acidshould be replicable either in vivo (e.g., as a vector) or in vitro(e.g., by PCR, transcription and translation). In vivo replication canbe autonomous (as for a cell), with the assistance of host factors (asfor a virus) or with the assistance of both host and helper virus (asfor a phagemid). Cells, spores or viruses are examples of displaypackages. The replicable genetic package can be eukaryotic orprokaryotic. A display library is formed by introducing nucleic acidsencoding exogenous polypeptides to be displayed into the genome of thedisplay package to form a fusion protein with an endogenous protein thatis normally expressed from the outer surface of the display package.Expression of the fusion protein, transport to the outer surface andassembly results in display of exogenous polypeptides from the outersurface of the genetic package.

A further type of display package comprises a polypeptide bound to anucleic acid encoding the polypeptide. Such an arrangement can beachieved in several ways. U.S. Pat. No. 5,733,731 describe a method inwhich a plasmid is engineered to expression a fusion protein comprisinga DNA binding polypeptide and a polypeptide to be screened. Afterexpression the fusion protein binds to the vector encoding it though theDNA binding polypeptide component. Vectors displaying fusion proteinsare screened for binding to a target, and vectors recovered for furtherrounds of screening or characterization. In another method, polypeptidesare screened as components of display package comprising a polypeptidebeing screened, and mRNA encoding the polypeptide, and a ribosomeholding together the mRNA and polypeptide (see Hanes & Pluckthun, PNAS94, 4937-4942 (1997); Hanes et al., PNAS 95, 14130-14135 (1998); Haneset al, FEBS Let. 450, 105-110 (1999); U.S. Pat. No. 5,922,545). mRNA ofselected complexes is amplified by reverse transcription and PCR and invitro transcription, and subject to further screening linked to aribosome and protein translated from the mRNA. In another method, RNA isfused to a polypeptide encoded by the RNA for screening (Roberts &Szostak, PNAS 94, 12297-12302 (1997), Nemoto et al., FEBS Letters 414,405-408 (1997). RNA from complexes surviving screening is amplified byreverse transcription PCR and in vitro transcription.

The genetic packages most frequently used for display libraries arebacteriophage, particularly filamentous phage, and especially phage M13,Fd and F1. Most work has inserted libraries encoding polypeptides to bedisplayed into either gIII or gVIII of these phage forming a fusionprotein. See, e.g., Dower, WO 91/19818; Devlin, WO 91/18989;MacCafferty, WO 92/01047 (gene III); Huse, WO 92/06204; Kang, WO92/18619 (gene VIII). Such a fusion protein comprises a signal sequence,usually from a secreted protein other than the phage coat protein, apolypeptide to be displayed and either the gene III or gene VIII proteinor a fragment thereof. Exogenous coding sequences are often inserted ator near the N-terminus of gene III or gene VIII although other insertionsites are possible. Some filamentous phage vectors have been engineeredto produce a second copy of either gene III or gene VIII. In suchvectors, exogenous sequences are inserted into only one of the twocopies. Expression of the other copy effectively dilutes the proportionof fusion protein incorporated into phage particles and can beadvantageous in reducing selection against polypeptides deleterious tophage growth. In another variation, exogenous polypeptide sequences arecloned into phagemid vectors which encode a phage coat protein and phagepackaging sequences but which are not capable of replication. Phagemidsare transfected into cells and packaged by infection with helper phage.Use of phagemid system also has the effect of diluting fusion proteinsformed from coat protein and displayed polypeptide with wild type copiesof coat protein expressed from the helper phage. See, e.g., Garrard, WO92/09690.

Eukaryotic viruses can be used to display polypeptides in an analogousmanner. For example, display of human heregulin fused to gp70 of Moloneymurine leukemia virus has been reported by Han, et al., Proc. Natl.Acad. Sci. USA 92:9747-9751 (1995). Spores can also be used as displaypackages. In this case, polypeptides are displayed from the outersurface of the spore. For example, spores from B. subtilis have beenreported to be suitable. Sequences of coat proteins of these spores areprovided by Donovan, et al., J. Mol. Biol. 196:1-10 (1987). Cells canalso be used as display packages. Polypeptides to be displayed areinserted into a gene encoding a cell protein that is expressed on thecells surface. Bacterial cells including Salmonella typhimurium,Bacillus subtilis, Pseudomonas aeruginosa, Vibrio cholerae, Klebsiellapneumonia, Neisseria gonorrhoeae, Neisseria meningitidis, Bacteroidesnodosus, Moraxella bovis, and especially Escherichia coli are preferred.Details of outer surface proteins are discussed by Ladner, et al., U.S.Pat. No. 5,571,698, and Georgiou, et al., Nature Biotechnology 15:29-34(1997) and references cited therein. For example, the lamB protein of E.coli is suitable.

2. Displayed Antibodies

Antibody chains can be displayed in single or double chain form. Singlechain antibody libraries can comprise the heavy or light chain of anantibody alone or the variable domain thereof. However, more typically,the members of single-chain antibody libraries are formed from a fusionof heavy and light chain variable domains separated by a peptide spacerwithin a single contiguous protein. See e.g., Ladner, et al., WO88/06630; McCafferty, et al., WO 92/01047. Double-chain antibodies areformed by noncovalent association of heavy and light chains or bindingfragments thereof. Double chain antibodies can also form by associationof two single chain antibodies, each single chain antibody comprising aheavy chain variable domain, a linker and a light chain variable domain.In such antibodies, known as diabodies, the heavy chain of onesingle-chain antibody binds to the light chain of the other and viceversa, thus forming two identical antigen binding sites (see Hollingeret al., Proc. Natl. Acad. Sci. USA 90, 6444-6448 (1993) and Carter &Merchan, Curr. Op. Biotech. 8, 449-454 (1997). Thus, phage displayingsingle chain antibodies can form diabodies by association of two singlechain antibodies as a diabody.

The diversity of antibody libraries can arise from obtainingantibody-encoding sequences from a natural source, such as a nonclonalpopulation of immunized or unimmunized B cells. Alternatively, oradditionally, diversity can be introduced by artificial mutagenesis ofnucleic acids encoding antibody chains before or after introduction intoa display vector. Such mutagenesis can occur in the course of PCR or canbe induced before or after PCR.

Nucleic acids encoding antibody chains to be displayed optionallyflanked by spacers are inserted into the genome of a display package asdiscussed above by standard recombinant DNA techniques (see generally,Sambrook, et al., Molecular Cloning, A Laboratory Manual, 2d ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989,incorporated by reference herein). The nucleic acids are ultimatelyexpressed as antibody chains (with or without spacer or frameworkresidues). In phage, bacterial and spore vectors, antibody chains arefused to all or part of the an outer surface protein of the replicablepackage. Libraries often have sizes of about 10³, 10⁴, 10⁶, 10⁷, 10⁸ ormore members.

Double-chain antibody display libraries represent a species of thedisplay libraries discussed above. Production of such libraries isdescribed by, e.g., Dower, U.S. Pat. No. 5,427,908; U.S. Pat. No.5,580,717, Huse WO 92/06204; Huse, in Antibody Engineering, (Freeman1992), Ch. 5; Kang, WO 92/18619; Winter, WO 92/20791; McCafferty, WO92/01047; Hoogenboom WO 93/06213; Winter, et al., Annu. Rev. Immunol.12:433-455 (1994); Hoogenboom, et al., Immunological Reviews 130:41-68(1992); Soderlind, et al., Immunological Reviews 130:109-124 (1992). Forexample, in double-chain antibody phage display libraries, one antibodychain is fused to a phage coat protein, as is the case in single chainlibraries. The partner antibody chain is complexed with the firstantibody chain, but the partner is not directly linked to a phage coatprotein. Either the heavy or light chain can be the chain fused to thecoat protein. Whichever chain is not fused to the coat protein is thepartner chain. This arrangement is typically achieved by incorporatingnucleic acid segments encoding one antibody chain gene into either gIIIor gVIII of a phage display vector to form a fusion protein comprising asignal sequence, an antibody chain, and a phage coat protein. Nucleicacid segments encoding the partner antibody chain can be inserted intothe same vector as those encoding the first antibody chain. Optionally,heavy and light chains can be inserted into the same display vectorlinked to the same promoter and transcribed as a polycistronic message.Alternatively, nucleic acids encoding the partner antibody chain can beinserted into a separate vector (which may or may not be a phagevector). In this case, the two vectors are expressed in the same cell(see WO 92/20791). The sequences encoding the partner chain are insertedsuch that the partner chain is linked to a signal sequence, but is notfused to a phage coat protein. Both antibody chains are expressed andexported to the periplasm of the cell where they assemble and areincorporated into phage particles.

Typically, only the variable region of human light and heavy chains arecloned from a nonhuman transgenic animal. In such instances, the displayvector can be designed to express heavy and light chain constant regionsor fragments thereof in-frame with heavy and light chain variableregions expressed from inserted sequences. Typically, the constantregions are naturally occurring human constant regions; a fewconservative substitutions can be tolerated but are not preferred. In aFab fragment, the heavy chain constant region usually comprises a C_(H)1region, and optionally, part or all of a hinge region, and the lightchain constant region is an intact light chain constant region, such asC_(κ) or C_(λ). Choice of constant region isotype depends in part onwhether complement-dependent cytotoxity is ultimately required. Forexample, human isotypes IgG1 and IgG4 support such cytotoxicity whereasIgG2 and IgG3 do not. Alternatively, the display vector can providenonhuman constant regions. In such situations, typically, only thevariable regions of antibody chains are subsequently subcloned fromdisplay vectors and human constant regions are provided by an expressionvector in frame with inserted antibody sequences.

In a further variation, both constant and variable regions can be clonedfrom the transgenic animal. For example, heavy chain variable regionscan be cloned linked to the C_(H)1 constant region and light chainvariable regions linked to an intact light chain constant region forexpression of Fab fragments. In this situation, display vectors need notencode constant regions.

Antibody encoding sequences can be obtained from lymphatic cells of anonhuman transgenic animal. Typically, the cells have been immunized, inwhich case immunization can be performed in vivo before harvestingcells, or in vitro after harvesting cells, or both. Spleen cells of animmunized animal are a preferred source material. Immunization can beperformed with any type of antigen. Antigens are often human proteins.

Rearranged immunoglobulin genes can be cloned from genomic DNA or mRNA.For the latter, mRNA is extracted from the cells and cDNA is preparedusing reverse transcriptase and poly dT oligonucleotide primers. Primersfor cloning antibody encoding sequences are discussed by Larrick, etal., Bio/Technology 7:934 (1989), Danielsson & Borrebaceick, in AntibodyEngineering: A Practical Guide (Freeman, N.Y., 1992), p. 89 and Huse,id. at Ch. 5.

Repertoires of antibody fragments have been constructed by combiningamplified V_(H) and V_(L) sequences together in several ways. Light andheavy chains can be inserted into different vectors and the vectorscombined in vitro (Hogrefe, et al., Gene 128:119-126 (1993)) or in vivo(Waterhouse, et al., Nucl. Acids. Res. :2265-66 (1993)). Alternatively,the light and heavy chains can be cloned sequentially into the samevector (Barbas, et al., Proc. Natl. Acad. Sci. USA 88: 7987-82 (1991))or assembled together by PCR and then inserted into a vector (Clackson,et al., Nature 352:624-28 (1991)). Repertoires of heavy chains can bealso be combined with a single light chain or vice versa. Hoogenboom, etal., J. Mol. Biol. 227: 381-88 (1992).

Typically, segments encoding heavy and light antibody chains aresubcloned from separate populations of heavy and light chains resultingin random association of a pair of heavy and light chains from thepopulations in each vector. Thus, modified vectors typically containcombinations of heavy and light chain variable region not found innaturally occurring antibodies. Some of these combinations typicallysurvive the selection process and also exist in the polyclonal librariesdescribed below.

Some exemplary vectors and procedures for cloning populations of heavychain and light chain encoding sequences have been described by Huse, WO92/06204. Diverse populations of sequences encoding H_(C) polypeptidesare cloned into M13IX30 and sequences encoding L_(C) polypeptides arecloned into M13IX11. The populations are inserted between the XhoI-SeeIor StuI restriction enzyme sites in M13IX30 and between the SacI-XbaI orEcoRV sites in M13IX11 (FIGS. 1A and B of Huse, respectively). Bothvectors contain two pairs of MluI-HindIII restriction enzyme sites(FIGS. 1A and B of Huse) for joining together the H_(C) and L_(C)encoding sequences and their associated vector sequences. The two pairsare symmetrically orientated about the cloning site so that only thevector proteins containing the sequences to be expressed are exactlycombined into a single vector.

Others exemplary vectors and procedures for cloning antibody chains intofilamentous phage are described in the present Examples.

D. Enrichment for Polyvalent Display Members

1. Theory of the Method

That a display library should preferably be enriched for membersdisplaying multiple copies of a polypeptide is a finding apparently atvariance with some early reports in the field. See, e.g., Cwirla et al.,supra. Most work on display libraries has been done by inserting nucleicacid libraries into pIII or pVIII of filamentous phage. Because pIII ispresent in 4 or 5 copies per phage and pVIII is present in severalhundred copies per phage, some early reports assumed that foreignpolypeptides would be displayed in corresponding numbers per phage.However, more recent work has made clear that the actual number ofcopies of polypeptide displayed per phage is well below theoreticalexpectations, perhaps due to proteolytic cleavage of polypeptides.Winter, et al., Ann. Rev. Immunol. 12:433-55 (1994). Further, vectorsystems used for phage display often encode two copies of a phage coatprotein, one of which is a wild type protein and the other of whichforms a fusion protein with exogenous polypeptides to be displayed. Bothcopies are expressed and the wild type coat protein effectively dilutesthe representation of the fusion protein in the phage coat.

A typical ratio of displayed Fabs per phage, when Fabs are expressedfrom pVIII of a filamentous phage is about 0.2. The probability, Pr(y),of y Fabs being expressed on a phage particle if the average frequencyof expression per phage is n is given by the Poisson probabilitydistribution

Pr(y)=e ^(−n) n ^(y) /y!

For a frequency of 0.2 Fabs per phage, the probabilities for theexpression of 0, 1, 2, and 3 Fabs per phage are 0.82, 0.16, 0.016, and0.0011. The proportion of phage particle displaying two or more Fabs istherefore only 0.017.

The low representation of members displaying more than one Fab fragmentin a phage display library can be related to the result that only asmall percentage of such library members are capable of survivingaffinity selection to immobilized binding partners. A library wasconstructed in which all members encoded the same Fab fragment which wasknown to have a high binding affinity for a particular target. It wasfound that even under the mildest separation conditions for removal offree from bound phage, it was not possible to bind more than about 0.004of the total phage. This proportion is the same order of magnitude asthe proportion of phage displaying at least two Fab fragments,suggesting that phage must display at least two Fab fragments to bind toimmobilized target. Probably shear forces dissociate phage displayingonly a single Fab fragment from the solid phase. Therefore, at least twobinding events are necessary for a phage-Fab library member to be boundto immobilized target with sufficient avidity to enable separation ofthe bound from the free phage. It is expected that similar constraintsapply in other forms of display library.

Therefore, a preferred strategy of the present invention is to enrichfor library members binding to a receptor fused to displayed antibodychains before the library is contacted with a screening target. It isbelieved that the prescreening enriches for library members displayingat least two copies of a tag and therefore at least two copies of anantibody chain linked to the tag. Library members lacking two or moreantibody chains, which are incapable of surviving affinity selection viabinding through displayed antibody chain to any immobilized screeningtarget, but which nevertheless can survive affinity selection byformation of multiple nonspecific bonds to such a target or its support,are thus substantially eliminated before screening of the library to thetarget is performed.

2. Tags and Receptors

The above strategy is effected by the use of paired tags and receptors.A tag can any peptide sequence that is common to different members ofthe library, heterologous to the display package, and fused to apolypeptide displayed from the display package. For example, a tag canbe a synthetic peptide sequence, a constant region of an antibody. Insome methods, single chain antibodies are displayed in which only thelight or heavy chain variable region but not both varies betweenmembers. In such situations, among others, the variable region that isthe same in different members can be used as a tag. Suitabletag-receptor combinations include epitope and antibody; for example,many high affinity hexapeptide ligands are known for the anti-dynorphinmAb 32.39, (see Barrett et al., Neuropeptides 6:113-120 (1985) and Cullet al., Proc. Nat'l Acad. Sci. USA 89:1865-1869 (1992)) and a variety ofshort peptides are known to bind the MAb 3E7 (Schatz, Biotechnology11:1138-43 (1993)). Another combination of tag and antibody is describedby Blanar & Rutter, Science 256:1014-1018 (1992).

Another example of a tag-receptor pair is the FLAG™ system (Kodak). TheFLAG™ molecular tag consists of an eight amino acid FLAG peptide markerthat is linked to the target binding moiety. A 24 base pair segmentcontaining a FLAG coding sequence can be inserted adjacent to anucleotide sequence that codes for the displayed polypeptide. The FLAGpeptide includes an enterokinase recognition site that corresponds tothe carboxyl-terminal five amino acids. Capture moieties suitable foruse with the FLAG peptide marker include antibodies Anti-FLAG M1, M2 andM5, which are commercially available.

Still other combinations of peptides and antibodies can be identified byconventional phage display methods. Further suitable combinations ofpeptide sequence and receptor include polyhistidine and metal chelateligands containing Ni²⁺ immobilized on agarose (see Hochuli in GeneticEngineering: Principles and Methods (ed. J K Setlow, Plenum Press,N.Y.), Ch. 18, pp. 87-96 and maltose binding protein (Maina, et al.,Gene 74:365-373 (1988)).

Receptors are often labeled with biotin allowing the receptors to beimmobilized to an avidin-coated support. Biotin labeling can beperformed using the biotinylating enzyme, BirA (see, e.g., Schatz,Biotechnology 11:1138-43 (1993)).

A nucleic acid sequence encoding a tag is inserted into a display vectorin such a manner that the tag is expressed as part of the fusion proteincontaining the polypeptide to be displayed and an outer surface proteinof the display package. The relative ordering of these components is notcritical provided that the tag and polypeptide to be displayed are bothexposed on the outer surface of the package. For example, the tag can beplaced between the outer surface protein and the displayed polypeptideor at or near the exposed end of the fusion protein.

In display packages displaying Fabs, a tag can be fused to either theheavy or the light Fab chain, irrespective which chain is linked to aphage coat protein. Optionally, two different tags can used one fused toeach of the heavy and light chains. One tag is usually positionedbetween the phage coat protein and antibody chain linked thereto, andthe other tag is positioned at either the N- or C-terminus of thepartner chain.

3. Selection of Polyvalent Library Members Members

Selection of polyvalent library members is performed by contacting thelibrary with the receptor for the tag component of library members.Usually, the library is contacted with the receptor immobilized to asolid phase and binding of library members through their tag to thereceptor is allowed to reach equilibrium. The complexed receptor andlibrary members are then brought out of solution by addition of a solidphase to which the receptor bears affinity (e.g., an avidin-labeledsolid phase can be used to immobilize biotin-labeled receptors).Alternatively, the library can be contacted with receptor in solutionand the receptor subsequently immobilized. The concentration of receptorshould usually be at or above the Kd of the tag/receptor during solutionphase binding so that most displayed tags bind to a receptor atequilibrium. When the receptor-library members are contacted with thesolid phase only the library members linked to receptor through at leasttwo displayed tags remain bound to the solid phase following separationof the solid phase from library members in solution. Library memberslinked to receptor through a single tag are presumably sheared from thesolid phase during separation and washing of the solid phase. Afterremoval of unbound library members, bound library members can bedissociated from the receptor and solid phase by a change in ionicstrength or pH, or addition of a substance that competes with the tagfor binding to the receptor. For example, binding of metal chelateligands immobilized on agarose and containing Ni²⁺ to a hexahistidinesequence is easily reversed by adding imidazole to the solution tocompete for binding of the metal chelate ligand. Antibody-peptidebinding can often be dissociated by raising the pH to 10.5 or higher.

The average number of polypeptides per library member selected by thismethod is affected by a number of factors. Decreasing the concentrationof receptor during solution-phase binding has the effect of increasingthe average number of polypeptides in selected library members. Anincrease in the stringency of the washing conditions also increases theaverage number of polypeptides per selected library member. The physicalrelationship between library members and the solid phase can also bemanipulated to increase the average number of polypeptides per librarymember. For example, if discrete particles are used as the solid phase,decreasing the size of the particles increases the steric constraints ofbinding and should require a higher density of polypeptides displayedper library member.

For Fab libraries having two tags, one linked to each antibody chain,two similar rounds of selection can be performed, with the products ofone round becoming the starting materials for the second round. Thefirst round of selection is performed with a receptor to the first tag,and the second round with a receptor to the second tag. Selecting forboth tags enriches for library members displaying two copies of bothheavy and light antibody chains (i.e., two Fab fragments).

Although the theory underlying the above methods of polyvalentenrichment is believed to be correct, the practice of the invention isin no way dependent on the correctness of this theory. Prescreening adisplay library for members binding to a tag, followed by screeningthose members for binding to a target results in a higher degree ofenrichment for members with affinity for a target than if the method isperformed without the prescreening step. Thus, the method can bepracticed as described, and achieve the desired result of highlyenriched libraries without any understanding of the underlyingmechanism.

4. Selection for Affinity to Target

Library members displaying antibody chains, with or without prescreeningto a tag receptor, are screened for binding to a target. The target canbe any molecule of interest for which it is desired to identify bindingpartners. The target should lack specific binding affinity for thetag(s) (if used), because in this step it is the displayed polypeptidesbeing screened, and not the tags that bind to the target. The screeningprocedure at this step is closely analogous to the prescreening stepexcept that the affinity reagent is a target of interest rather than areceptor to a tag. The enriched library members are contacted with thetarget which is usually labeled (e.g., with biotin) in such a mannerthat allows its immobilization. Binding is allowed to proceed toequilibrium and then target is brought out of solution by contactingwith the solid phase in a process known as panning (Parmley & Smith,Gene 73:305-318 (1988)). Library members that remain bound to the solidphase throughout the selection process do so by virtue of polyvalentbonds between them and immobilized target molecules. Unbound librarymembers are washed away from the solid phase. In some methods, librarymembers are screened by binding to cells displaying a receptor ofinterest. Thereafter, the entire cell population can be recovered bycentrifugation or fractions bound to phage can be isolated by labellingwith a phage specific antibody and separating labelled phage bound tocells using magnetic beads or FACS™.

Usually, library members are subject to amplification before performinga subsequent round of screening. Often, bound library members can beamplified without dissociating them from the support. For example, geneVIII phage library members immobilized to beads, can be amplified byimmersing the beads in a culture of E. coli. Likewise, bacterial displaylibraries can be amplified by adding growth media to bound librarymembers. Alternatively, bound library members can be dissociated fromthe solid phase (e.g., by change of ionic strength or pH) beforeperforming subsequent selection, amplification or propagation.

After affinity selection, bound library members are now enriched forantibody chains having specific affinity for the target of interest (andfor polyvalent display members if a prescreening step has beenperformed). After subsequent amplification, to produce a secondarylibrary, the secondary library remains enriched for display ofpolypeptides having specific affinity for the target, but, as a resultof amplification, is no longer enriched for polyvalent display ofpolypeptides. Thus, a second cycle of polyvalent enrichment can then beperformed, followed by a second cycle of affinity enrichment to thescreening target. Further cycles of affinity enrichment to the screeningtarget, optionally, alternating with amplification and enrichment forpolyvalent display can then be performed, until a desired degree ofenrichment has been achieved.

In a variation, affinity screening to a target is performed incompetition with a compound that resembles but is not identical to thetarget. Such screening preferentially selects for library members thatbind to a target epitope not present on the compound. In a furthervariation, bound library members can be dissociated from the solid phasein competition with a compound having known crossreactivity with atarget for an antigen. Library members having the same or similarbinding specificity as the known compound relative to the target arepreferentially eluted. Library members with affinity for the targetthrough an epitope distinct from that recognized by the compound remainbound to the solid phase.

Discrimination in selecting between antibody chains of differentmonovalent affinities for the target is affected by the valency oflibrary members and the concentration of target during the solutionphase binding. Assuming a minimum of i labeled target molecules must bebound to a library member to immobilize it on a solid phase, then theprobability of immobilization can be calculated for a library memberdisplaying n polypeptides. From the law of mass action, the bound/totalantibody chain fraction, F, is K[targ]/(1+K[targ]), where [targ] is thetotal target concentration in solution. Thus, the probability that i ormore displayed antibody chains per library member are bound by thelabeled target is given by the binomial probability distribution:$\sum\limits_{n}^{y = i}\quad \left( {{{n!}/\left\lbrack {{y!}{\left( {n - y} \right)!}} \right\rbrack}{F^{y}\left( {1 - F} \right)}^{n - y}} \right.$

As the probability is a function of K and [target], multivalent displaymembers each having a monovalent affinity, K, for the target can beselected by varying the concentration of target. The probabilities ofsolid-phase immobilization for i=1, 2, or 3, with library membersexhibiting monovalent affinities of 0.1/[Ag], 1/[Ag], and 10/[Ag], anddisplaying n polypeptides per member are:

n K = 0.1/[targ] K = 1/[targ] K = 10/[targ] Probability ofImmobilization (i = 1) 1 0.09 0.5 0.91 2 0.17 0.75 0.99 3 0.25 0.875 40.32 0.94 5 0.38 0.97 6 0.44 0.98 7 0.49 0.99 8 0.53 9 0.58 10 0.61 200.85 50 0.99 Probability of Immobilization (i = 2) 2 0.008 0.25 0.83 30.023 0.50 0.977 4 0.043 0.69 0.997 5 0.069 0.81 6 0.097 0.89 7 0.1280.94 8 0.160 0.965 9 0.194 0.98 20 0.55 50 0.95 Probability ofImmobilization (i = 3) 3 0.00075 0.125 0.75 4 0.0028 0.31 0.96 5 0.00650.50 0.99 6 0.012 0.66 7 0.02 0.77 8 0.03 0.855 9 0.0415 0.91 10 0.0550.945 12 0.089 0.98 14 0.128 0.99 20 0.27 50 0.84

The above tables show that the discrimination between immobilizingpolypeptides of different monovalent binding affinities is affected bythe valency of library members (n) and by the concentration of targetfor the solution binding phase. Discrimination is maximized when n(number of polypeptides displayed per phage) is equal to i (minimumvalency required for solid phase binding). Discrimination is alsoincreased by lowering the concentration of target during the solutionphase binding. Usually, the target concentration is around the Kd of thepolypeptides sought to be isolated. Target concentration of 10⁻⁸-10⁻¹⁰ Mare typical.

Enriched libraries produced by the above methods are characterized by ahigh proportion of members encoding polypeptides having specificaffinity for the target. For example, at least 10, 25, 50, 75, 80, 90,95, or 99% of members encode polypeptides having specific affinity forthe target. In some libraries, at least 10, 25, 50, 75, 80, 90, 95, or99% of members have affinities of at least 10⁸ M⁻¹, 10⁹ M⁻¹ or 10¹⁰ M⁻¹.In libraries of double chain antibodies, a pair of segments encodingheavy and light chains of an antibody is considered a library member.The exact percentage of members having affinity for the target dependswhether the library has been amplified following selection, becauseamplification increases the representation of genetic deletions.However, among members with full-length polypeptide coding sequences,the proportion encoding polypeptides with specific affinity for thetarget is very high (e.g., at 50, 75, 80, 90, 95 or 99% having affinityof 10⁸ M⁻¹, 10⁹ M⁻¹ or 10¹⁰ M⁻¹. Not all the library members that encodean antibody chain with specific affinity for the target necessarilydisplay the antibody chain. For example, in a library in which 95% ofmembers with full-length coding sequences encode antibody chains withspecific affinity for the target, usually fewer than half actuallydisplay the antibody chain. Usually, such libraries have at least 4, 10,20, 50, 100, 1000, 10,000 or 100,000 different coding sequences.Usually, the representation of any one such coding sequences is no morethan 50%, 25% or 10% of the total coding sequences in the library.

F. Subcloning Antibody Chains into an Expression Vector

Screening of display library members typically results in asubpopulation of library members having specific affinity for a target.There are a number of options at this point. In some methods, clonalisolates of library members are obtained, and these isolates useddirectly. In other methods, clonal isolates of library member areobtained, and DNA encoding antibody chains amplified from each isolate.Typically, heavy and light chains are amplified as components of thesame DNA molecule before transfer to an expression vector, such thatcombinations of heavy and light chain existing in the display vector arepreserved in the expression vector. For displayed antibody chains thatinclude both human variable regions and human constant regions,typically nucleic acids encoding both the variable region and constantregion are subcloned. In other methods, nucleic acids encoding antibodychains are amplified and subcloned en masse from a pool of librarymembers into multiple copies of an expression vector without clonalisolation of individual members.

The subcloning process is now described in detail for transfer of amixed population of nucleic acids from a display vector to an expressionvector. Essentially the same process can be used on nucleic acidsobtained from a clonal isolate of an individual display vector.

Nucleic acids encoding antibody chains to be subcloned can be excised byrestriction digestion of flanking sequences or can be amplified by PCRusing primers to sites flanking the coding sequences. See generally PCRTechnology: Principles and Applications for DNA Amplification (ed. H. A.Erlich, Freeman Press, NY, N.Y., 1992); PCR Protocols: A Guide toMethods and Applications (eds. Innis, et al., Academic Press, San Diego,Calif., 1990); Mattila, et al., Nucleic Acids Res. 19:967 (1991);Eckert, et al., PCR Methods and Applications 1:17 (1991); PCR (eds.McPherson et al., IRL Press, Oxford). PCR primers can contain a markersequence that allows positive selection of amplified fragments whenintroduced into an expression vector. PCR primers can also containrestriction sites to allow cloning into an expression vector, althoughthis is not necessary. For Fab libraries, if heavy and light chains areinserted adjacent or proximate to each other in a display vector, thetwo chains can be amplified or excised together. For some Fab libraries,only the variable domains of antibody chain(s) are excised or amplified.If the heavy or light chains of a Fab library are excised or amplifiedseparately, they can subsequently be inserted into the same or differentexpression vectors.

Having excised or amplified fragments encoding displayed antibodychains, the fragments are usually size-purified on an agarose gel orsucrose gradient. Typically, the fragments run as a single sharpfull-length band with a smear at lower molecular corresponding tovarious deleted forms of coding sequence. The band corresponding tofull-length coding sequences is removed from the gel or gradient andthese sequences are used in subsequent steps.

The next step is to join the nucleic acids encoding full-length codingsequences to an expression vector thereby creating a population ofmodified forms of the expression vector bearing different inserts. Thiscan be done by conventional ligation of cleaved expression vector with amixture of inserts cleaved to have compatible ends. Alternatively, theuse of restriction enzymes on insert DNA can be avoided. This method ofcloning is beneficial because naturally encoded restriction enzyme sitesmay be present within insert sequences, thus, causing destruction of thesequence when treated with a restriction enzyme. For cloning withoutrestricting, a mixed population of inserts and linearized vectorsequences are treated briefly with a 3′ to 5′ exonuclease such as T4 DNApolymerase or exonuclease III. See Sambrook, et al., Molecular Cloning,A Laboratory Manual (2nd Ed., CSHP, New York 1989). The protruding 5′termini of the insert generated by digestion are complementary tosingle-stranded overhangs generated by digestion of the vector. Theoverhangs are annealed, and the re-annealed vector transfected intorecipient host cells. The same result can be accomplished using 5′ to 3′exonucleases rather than a 3′ to 5′ exonuclease.

Preferably, ligation of inserts to expression vector is performed underconditions that allow selection against re-annealed vector and uncutvector. A number of vectors containing conditional lethal genes thatallow selection against re-annealed vector under nonpermissiveconditions are known. See, e.g., Conley & Saunders, Mol. Gen. Genet.194:211-218 (1984). These vectors effectively allow positive selectionfor vectors having received inserts. Selection can also be accomplishedby cleaving an expression vector in such a way that a portion of apositive selection marker (e.g., antibiotic resistance) is deleted. Themissing portion is then supplied by full-length inserts. The portion canbe introduced at the 3′ end of polypeptide coding sequences in thedisplay vector, or can be included in a primer used for amplification ofthe insert. An exemplary selection scheme, in which inserts supply aportion of a tetracycline-resistance gene promoter deleted by HindIIIcleavage of a pBR-derivative vector, is described in Example 14.

The choice of expression vector depends on the intended host cells inwhich the vector is to be expressed. Typically, the vector includes apromoter and other regulatory sequences in operable linkage to theinserted coding sequences that ensure the expression of the latter. Useof an inducible promoter is advantageous to prevent expression ofinserted sequences except under inducing conditions. Inducible promotersinclude arabinose, lacZ, metallothionein promoter or a heat shockpromoter. Cultures of transformed organisms can be expanded undernoninducing conditions without biasing the population for codingsequences whose expression products are better tolerated by the hostcells. The vector may also provide a secretion signal sequence positionto form a fusion protein with polypeptides encoded by insertedsequences, although often inserted polypeptides are linked to a signalsequences before inclusion in the vector. Vectors to be used to receivesequences encoding antibody light and heavy chain variable domainssometimes encode constant regions or parts thereof that can be expressedas fusion proteins with inserted chains thereby leading to production ofintact antibodies or fragments thereof. Typically, such constant regionsare human. Conservative mutations although not preferred can betolerated. For example, if display packages display a heavy chainvariable region linked to a C_(H)1 constant region and a light chainvariable region linked to an intact light chain constant region, and thecomplete antibody chains are transferred from the display vector to theexpression vector, then the expression vector can be designed to encodehuman heavy chain constant region hinge, C_(H)2 and C_(H)3 regionsin-frame with the C_(H)1 region of the inserted heavy chain nucleic acidthereby resulting in expression of an intact antibody. Of course, manyminor variations are possible as to precisely which segment of the humanheavy chain constant region is supplied by the display package and whichby the expression vector. For example, the display package can bedesigned to include a C_(H)1 region, and some or all of the hingeregion. In this case, the expression vector is designed to supply theresidual portion of the hinge region (if any) and the C_(H)2 and C_(H)3regions for expression of intact antibodies.

E. coli is one prokaryotic host useful particularly for cloning thepolynucleotides of the present invention. Other microbial hosts suitablefor use include bacilli, such as Bacillus subtilis, and otherenterobacteriaceae, such as Salmonella, Serratia, and variousPseudomonas species. In these prokaryotic hosts, one can also makeexpression vectors, which typically contain expression control sequencescompatible with the host cell (e.g., an origin of replication). Inaddition, any number of a variety of well-known promoters will bepresent, such as the lactose promoter system, a tryptophan (trp)promoter system, a beta-lactamase promoter system, or a promoter systemfrom phage lambda. The promoters typically control expression,optionally with an operator sequence, and have ribosome binding sitesequences and the like, for initiating and completing transcription andtranslation.

Other microbes, such as yeast, are also used for expression.Saccharomyces is a preferred host, with suitable vectors havingexpression control sequences, such as promoters, including3-phosphoglycerate kinase or other glycolytic enzymes, and an origin ofreplication, termination sequences and the like as desired. Insect cellsin combination with baculovirus vectors can also be used.

Mammalian tissue cell culture can also be used to express and producethe polypeptides of the present invention (see Winnacker, From Genes toClones (VCH Publishers, N.Y., N.Y., 1987). A number of suitable hostcell lines capable of secreting intact immunoglobulins have beendeveloped including the CHO cell lines, various Cos cell lines, HeLacells, myeloma cell lines, transformed B-cells and hybridomas.Expression vectors for these cells can include expression controlsequences, such as an origin of replication, a promoter, and an enhancer(Queen, et al., Immunol. Rev. 89:49-68 (1986)), and necessary processinginformation sites, such as ribosome binding sites, RNA splice sites,polyadenylation sites, and transcriptional terminator sequences.Preferred expression control sequences are promoters derived fromimmunoglobulin genes, SV40, adenovirus, bovine papilloma virus, orcytomegalovirus.

Methods for introducing vectors containing the polynucleotide sequencesof interest vary depending on the type of cellular host. For example,calcium chloride transfection is commonly utilized for prokaryoticcells, whereas calcium phosphate treatment or electroporation may beused for other cellular hosts. (See generally Sambrook, et al., supra).

Once expressed, collections of antibodies are purified from culturemedia and host cells. Usually, antibody chains are expressed with signalsequences and are thus released to the culture media. However, ifantibody chains are not naturally secreted by host cells, the antibodychains can be released by treatment with mild detergent. Antibody chainscan then be purified by conventional methods including ammonium sulfateprecipitation, affinity chromatography to immobilized target, columnchromatography, gel electrophoresis and the like (see generally Scopes,Protein Purification (Springer-Verlag, N.Y., 1982)).

The above methods result in novel libraries of nucleic acid sequencesencoding antibody chains having specific affinity for a chosen target.The libraries of nucleic acids typically have at least 5, 10, 20, 50,100, 1000, 10⁴ or 10⁵ different members. Usually, no single memberconstitutes more than 25 or 50% of the total sequences in the library.Typically, at least 25, 50%, 75, 90, 95, 99 or 99.9% of library membersencode antibody chains with specific affinity for the target molecules.In the case of double chain antibody libraries, a pair of nucleic acidsegments encoding heavy and light chains respectively is considered alibrary member. The nucleic acid libraries can exist in free form, ascomponents of any vector or transfected as a component of a vector intohost cells.

The nucleic acid libraries can be expressed to generate polyclonallibraries of antibodies having specific affinity for a target. Thecomposition of such libraries is determined from the composition of thenucleotide libraries. Thus, such libraries typically have at least 5,10, 20, 50, 100, 1000, 10⁴ or 10⁵ members with different amino acidcomposition. Usually, no single member constitutes more than 25 or 50%of the total polypeptides in the library. The percentage of antibodychains in an antibody chain library having specific affinity for atarget is typically lower than the percentage of corresponding nucleicacids encoding the antibody chains. The difference is due to the factthat not all polypeptides fold into a structure appropriate for bindingdespite having the appropriate primary amino acid sequence to supportappropriate folding. In some libraries, at least 25, 50, 75, 90, 95, 99or 99.9% of antibody chains have specific affinity for the targetmolecules. Again, in libraries of multi-chain antibodies, each antibody(such as a Fab or intact antibody) is considered a library member. Thedifferent antibody chains differ from each other in terms of finebinding specificity and affinity for the target. Some such librariescomprise members binding to different epitopes on the same antigen. Somesuch libraries comprises at least two members that bind to the sameantigen without competing with each other.

Polyclonal libraries of human antibodies resulting from the abovemethods are distinguished from natural populations of human antibodiesboth by the high percentages of high affinity binders in the presentlibraries, and in that the present libraries typically do not show thesame diversity of antibodies present in natural populations. The reduceddiversity in the present libraries is due to the nonhuman transgenicanimals that provide the source materials not including all humanimmunoglobulin genes. For example, some polyclonal antibody librariesare free of antibodies having lambda light chains. Some polyclonalantibody libraries of the invention have antibody heavy chains encodedby fewer than 10, 20, 30 or 40 V_(H) genes. Some polyclonal antibodylibraries of the invention have antibody light chains encoded by fewerthan 10, 20, 30 or 40 V_(L) genes.

III. Diagnostic Uses

1. Characteristics of Human Antibodies for Use as Diagnostic Reagents

Human antibodies for use in diagnostic methods of the invention arepreferably produced using the methods described above. The methodsresult in virtually unlimited numbers of human antibodies of any epitopebinding specificity and very high binding affinity to any desiredantigen. In general, the higher the binding affinity of an antibody forits target, the more stringent wash conditions can be performed in animmunoassay to remove nonspecifically bound material without removingtarget antigen. According, human antibodies useed in the above assaysusually have binding affinities of at least 10⁸, 10⁹, 10¹⁰, 10¹¹ or 10¹²M⁻¹. Further, it is desirable that antibodies used as diagnosticreagents have a sufficient on-rate to reach equilibrium under standardconditions such as those described in Example 28 in at least 12 hours,preferably at least five hours and more preferably at least one hour.

Human antibodies used in the claimed methods preferably have a highimmunoreactivity, that is, percentages of antibodies molecules that arecorrectly folded so that they can specifically bind their targetantigen. Such can be achieved by expression of sequences encoding theantibodies in E. coli as described above. Such expression usuallyresults in immunoreactivity of at least 80%, 90%, 95% or 99% (see Table4).

Some methods of the invention employ polyclonal preparations of humanantibodies as diagnostic reagents, and other methods employ monoclonalisolates. The use of polyclonal mixtures has a number of advantages withrespect to compositions made of one monoclonal antibody. By binding tomultiple sites on a target, polyclonal antibodies or other polypeptidescan generate a stronger signal (for diagnostics) than a monoclonal thatbinds to a single site. Further, a polyclonal preparation can bind tonumerous variants of a prototypical target sequence (e.g., allelicvariants, species variants, strain variants, drug-induced escapevariants) whereas a monoclonal antibody may bind only to theprototypical sequence or a narrower range of variants thereto. However,monoclonal antibodies are advantageous for detecting a single antigen inthe presence or potential presence of closely related antigens.

In methods employing polyclonal human antibodies prepared in accordancewith the methods described above, the preparation typically contains anassortment of antibodies with different epitope specificities to theintended target antigen. Such can be verified by the methods describedin Example 26. In some methods employing monoclonal antibodies, it isdesirable to have two antibodies of different epitope bindingspecificities. A difference in epitope binding specificities can bedetermined by a competition assay.

2. Samples and Target

Although human antibodies can be used as diagnostic reagents for anykind of sample, they are most useful as diagnostic reagents for humansamples. Samples can be obtained from any tissue or body fluid of apatient. Preferred sources of samples include, whole blood, plasma,semen, saliva, tears, urine, fecal material, sweat, buccal, skin andhair. Samples can also be obtained from biopsies of internal organs orfrom cancers. Samples can be obtained from clinical patients fordiagnosis or research or can be obtained from undiseased individuals, ascontrols or for basic research.

The methods can be used for detecting any type of target antigen.Exemplary target antigens including bacterial, fungal and viralpathogens that cause human disease, such as. IV, Hepatitis (A, B, & C),Influenza, Herpes, Giardia, Malaria, Leishmania, Staphylococcus Aureus,Pseudomonas aeruginosa. Other target antigens are human proteins whoseexpression levels or compositions have been correlated with humandisease or other phenotype. Examples of such antigens include adhesionproteins, hormones, growth factors, cellular receptors, autoantigens,autoantibodies, and amyloid deposits. Other targets of interest includetumor cell antigens, such as carcinoembryonic antigen. Other antigens ofinterest are class I and class II MHC antigens.

3. HAMA and Heterophilic Antibodies

At least some human samples contain human antibodies that specificallybind to antibodies from a different species. Some such human antibodiesbind to the isotype of antibodies from a nonhuman species, and otherhuman antibodies bind to the idiotype of antibodies from a nonhumanspecies. Such human antibodies can arise by a variety of mechanisms. Forexample, administration of a mouse antibody (such as FDA-approved OKT3)to a human patient typically generates a human antimouse response. Asimilar response can be generated by environmental exposure to mouseantigens. Human antibodies that specifically bind to antibodies fromother species, such as rabbit or bovine, can likewise be generated byenvironmental exposure to antigens from rabbit or bovine. Further,exposure of humans to certain viruses, particularly Epstein Barr virus,the agent responsible for infectious mononucleosis generates a class ofantibodies termed heterophilic antibodies that bind to antibodies fromnonhuman species.

The frequency of human antibodies reactive with antibodies from nonhumanspecies in human patient samples has been the subject of varyingreports. For example, estimates of frequency of human anti-mouse IgGvary from 0.72% to 80% in different studies, and estimates of humananti-rabbit IgG have varied from 0.09% to 5% (see Kricka et al.,Clinical Chemistry 45, 942-956 (1999)). Regardless of the precisefrequency, there is clearly a significant risk that any human samplecontains human antibodies reactive with antibodies from some nonhumananimal. Therefore, use of an antibody from a nonhuman species as adiagnostic reagent runs a risk of generating inaccurate results.Inaccuracies can be reduced but not eliminated by using chimericantibodies. Some inaccuracies remain due to the presence of humanantibodies binding to the idiotype of the chimeric antibodies. Theinaccuracies are however eliminated by the use of human antibodies asdiagnostic reagents. There are no antibodies present in a typical humansample that bind to fully human antibodies.

4. Formats for Diagnostic Assays

Human antibodies can be used to detect a given target in a variety ofstandard assay formats. Such formats include immunoprecipitation,Western blotting, ELISA, radioimmunoassay, and immunometric assays. SeeHarlow & Lane, Antibodies, A Laboratory Manual (CSHP NY, 1988); U.S.Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,879,262; 4,034,074,3,791,932; 3,817,837; 3,839,153; 3,850,752; 3,850,578; 3,853,987;3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345;4,034,074; and 4,098,876.

Immunometric or sandwich assays are a preferred format (see U.S. Pat.No. 4,376,110, 4,486,530, 5,914,241, and 5,965,375). Such assays use oneantibody or population of antibodies immobilized to a solid phase, andanother antibody or population of antibodies in solution. Typically, thesolution antibody or population of antibodies is labelled. If anantibody population is used, the population typically containsantibodies binding to different epitope specificities within the targetantigen. Accordingly, the same population can be used for both solidphase and solution antibody. If monoclonal antibodies are used, firstand second monoclonal antibodies having different binding specificitiesare used for the solid and solution phase. Solid phase and solutionantibodies can be contacted with target antigen in either order orsimultaneously. If the solid phase antibody is contacted first, theassay is referred to as being a forward assay. Conversely, if thesolution antibody is contacted first, the assay is referred to as beinga reverse assay. If target is contacted with both antibodiessimultaneously, the assay is referred to as a simultaneous assay. Aftercontacting the target with antibody, a sample is incubated for a periodthat usually varies from about 10 min to about 24 hr and is usuallyabout 1 hr. A wash step is then performed to remove components of thesample not specifically bound to the antibody(ies) being used as adiagnostic reagent. When solid phase and solution antibodies are boundin separate steps, a wash can be performed after either or both bindingsteps. After washing, binding is quantified, typically by detectinglabel linked to the solid phase through binding of labelled solutionantibody. Usually for a given pair of antibodies or populations ofantibodies and given reaction conditions, a calibration curve isprepared from samples containing known concentrations of target antigen.Concentrations of antigen in samples being tested are then read byinterpolation from the calibration curve. Analyte can be measured eitherfrom the amount of labelled solution antibody bound at equilibrium or bykinetic measurements of bound labelled solution antibody at a series oftime points before equilibrium is reached. The slope of such a curve isa measure of the concentration of target in a sample

Suitable detectable labels for use in the above methods include anymoiety that is detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical, chemical, or other means. Forexample, suitable labels include biotin for staining with labeledstreptavidin conjugate, fluorescent dyes (e.g., fluorescein, Texas red,rhodamine, green fluorescent protein, and the like), radiolabels (e.g.,.sup.3 H, .sup.125 I, .sup.35 S, .sup.14 C, or .sup.32 P), enzymes(e.g., horseradish peroxidase, alkaline phosphatase and others commonlyused in an ELISA), and colorimetric labels such as colloidal gold orcolored glass or plastic (e.g., polystyrene, polypropylene, latexbeads). Patents that described the use of such labels include U.S. Pat.Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149;and 4,366,241. See also Handbook of Fluorescent Probes and ResearchChemicals (6th Ed., Molecular Probes, Inc., Eugene Oreg.). Radiolabelscan be detected using photographic film or scintillation counters,fluorescent markers can be detected using a photodetector to detectemitted light. Enzymatic labels are typically detected by providing theenzyme with a substrate and detecting the reaction product produced bythe action of the enzyme on the substrate, and colorimetric labels aredetected by simply visualizing the colored label.

Suitable supports for use in the above methods include, for example,nitrocellulose membranes, nylon membranes, and derivatized nylonmembranes, and also particles, such as agarose, a dextran-based gel,dipsticks, particulates, microspheres, magnetic particles, test tubes,microtiter wells, SEPHADEX.™. (Amersham Pharmacia Biotech, PiscatawayN.J., and the like. Immobilization can be by absorption or by covalentattachment. Optionally, antibodies can be joined to a linker molecule,such as biotin for attachment to a surface bound linker, such as avidin.

Although the invention has been described in detail for purposes ofclarity of understanding, it will be obvious that certain modificationsmay be practiced within the scope of the appended claims. Allpublications and patent documents cited in this application are herebyincorporated by reference in their entirety for all purposes to the sameextent as if each were so individually denoted. A cell lines producing7F11 (HB-12443, Dec. 5, 1997) has been deposited at the American TypeCulture Collection, Rockville, Md. under the Budapest Treaty on thedates indicated and given the accession numbers indicated. The depositswill be maintained at an authorized depository and replaced in the eventof mutation, nonviability or destruction for a period of at least fiveyears after the most recent request for release of a sample was receivedby the depository, for a period of at least thirty years after the dateof the deposit, or during the enforceable life of the related patent,whichever period is longest. All restrictions on the availability to thepublic of these cell lines will be irrevocably removed upon the issuanceof a patent from the application.

EXAMPLE 1 Purification of RNA from Mouse Spleens

Mice having 3 different sets of human heavy chain genes were used tomake the antibody phage libraries to interleukin 8. Production of miceis described in Examples 23 and 24. The mice were immunized withinterleukin 8 (Example 19). Mice were immunized with 25 microgram ofantigen at 0.713 mg/ml. In a first procedure, mice were immunized once amonth beginning with CFA followed by IFA until a high human gamma titerwas reached (ca 6500) after a further six weeks, mice were boosted ip ondays -7, -6, -5, and sacrificed 5 days later. In an alternativeprocedure, mice were immunized every two weeks beginning with CFA andfollowed by IFA. After a high human gamma titer was reached, mice wereboosted on days -3, and -2 and sacrificed two days later.

The spleens were harvested in a laminar flow hood and transferred to apetri dish, trimming off and discarding fat and connective tissue. Thespleen was, working quickly, macerated with the plunger from a sterile 5cc syringe in the presence of 1.0 ml of solution D (25.0 g guanidinethiocyanate (Roche Molecular Biochemicals, Indianapolis, Ind.), 29.3 mlsterile water, 1.76 ml 0.75 M sodium citrate (pH 7.0), 2.64 ml 10%sarkosyl (Fisher Scientific, Pittsburgh, Pa.), 0.36 ml 2-mercaptoethanol(Fisher Scientific, Pittsburgh, Pa.)). The spleen suspension was pulledthrough an 18-gauge needle until viscous and all cells were lysed, thentransferred to a microcentrifuge tube. The petri dish was washed with100 μl of solution D to recover any remaining spleen, and this wastransferred to the tube. The suspension was then pulled through a22-gauge needle an additional 5-10 times. The sample was divided evenlybetween two microcentrifuge tubes and the following added in order, withmixing by inversion after each addition: 100 μl 2 M sodium acetate (pH4.0), 1.0 ml water-saturated phenol (Fisher Scientific, Pittsburgh,Pa.), 200 μl chloroform/isoamyl alcohol 49:1 (Fisher Scientific,Pittsburgh, Pa.). The solution was vortexed for 10 seconds and incubatedon ice for 15 min. Following centrifugation at 14 krpm for 20 min at2-8° C., the aqueous phase was transferred to a fresh tube. An equalvolume of water saturated phenol/chloroform/isoamyl alcohol (50:49:1)was added, and the tube was vortexed for ten seconds. After a 15 minincubation on ice, the sample was centrifuged for 20 min at 2-8° C., andthe aqueous phase was transferred to a fresh tube and precipitated withan equal volume of isopropanol at −20° C. for a minimum of 30 min.Following centrifugation at 14,000 rpm for 20 min at 4° C., thesupernatant was aspirated away, the tubes briefly spun and all traces ofliquid removed. The RNA pellets were each dissolved in 300 μl ofsolution D, combined, and precipitated with an equal volume ofisopropanol at −20° C. for a minimum of 30 min. The sample wascentrifuged 14,000 rpm for 20 min at 4° C., the supernatant aspirated asbefore, and the sample rinsed with 100 μl of ice-cold 70% ethanol. Thesample was again centrifuged 14,000 rpm for 20 min at 4° C., the 70%ethanol solution aspirated, and the RNA pellet dried in vacuo. Thepellet was resuspended in 100 μl of sterile distilled water. Theconcentration was determined by A₂₆₀ using an absorbance of 1.0 for aconcentration of 40 μg/ml. The RNA was stored at −80° C.

EXAMPLE 2 Preparation of Complementary DNA (cDNA)

The total RNA purified as described above was used directly as templatefor cDNA. RNA (50 μg) was diluted to 100 μL with sterile water, and 10μL-130 ng/μL oligo dT12 (synthesized on Applied Biosystems Model 392 DNAsynthesizer at Biosite Diagnostics) was added. The sample was heated for10 min at 70° C., then cooled on ice. 40 μL 5×first strand buffer wasadded (Gibco/BRL, Gaithersburg, Md.), 20 μL 0.1 M dithiothreitol(Gibco/BRL, Gaithersburg, Md.), 10 μL 20 mM deoxynucleosidetriphosphates (dNTP's, Roche Molecular Biochemicals, Indianapolis,Ind.), and 10 μL water on ice. The sample was then incubated at 37° C.for 2 min. 10 μL reverse transcriptase (Superscript™ II, Gibco/BRL,Gaithersburg, Md.) was added and incubation was continued at 37° C. for1 hr. The cDNA products were used directly for polymerase chain reaction(PCR).

EXAMPLE 3 Amplification of Human Antibody Sequence cDNA by PCR

The cDNA of four mice having the genotype HCo7 was amplified using 3-5′oligonucleotides and 1-3′ oligonucleotide for heavy chain sequences(Table A), and 10-5′ oligonucleotides and 1-3′ oligonucleotide for thekappa chain sequences (Table B). The cDNA of one mouse having thegenotype HCo12 was amplified using 5-5′ oligonucleotides and 1-3′oligonucleotide for heavy chain sequences (Table C), and theoligonucleotides shown in Table B for the kappa chain sequences. ThecDNA of two mice having the genotype HCo7/Co12 was amplified using theoligonucleotide sequences shown in Tables A and C for the heavy chainsequences and oligonucleotides shown in Table B for the kappa chainsequences. The 5′ primers were made so that a 20 nucleotide sequencecomplementary to the M13 uracil template was synthesized on the 5′ sideof each primer. This sequence is different between the H and L chainprimers, corresponding to 20 nucleotides on the 3′ side of the pelBsignal sequence for L chain primers and the alkaline phosphatase signalsequence for H chain primers. The constant region nucleotide sequencesrequired only one 3′ primer each to the H chains and the kappa L chains(Tables A and B). Amplification by PCR was performed separately for eachpair of 5′ and 3′ primers. A 50 μL reaction was performed for eachprimer pair with 50 pmol of 5′ primer, 50 pmol of 3′ primer, 0.25 μL TaqDNA Polymerase (5 units/μL, Roche Molecular Biochemicals, Indianapolis,Ind.), 3 μL cDNA (described in Example 2), 5 μL 2 mM dNTP's, 5 μL 10×TaqDNA polymerase buffer with MgCl₂ (Roche Molecular Biochemicals,Indianapolis, Ind.), and H₂O to 50 μL. Amplification was done using aGeneAmp® 9600 thermal cycler (Perkin Elmer, Foster City, Calif.) withthe following program: 94° C. for 1 min; 30 cycles of 94° C. for 20 sec,55° C. for 30 sec, and 72° C. for 30 sec; 72° C. for 6 min; 4° C.

Table A. Heavy chain oligonucleotides used to amplify cDNA for Hco7mice. Oligonucleotides 188 (SEQ ID NO:1), 944 (SEQ ID NO:2) and 948 (SEQID NO: 3) are 5′ primers and oligonucleotide 952 (SEQ ID NO:4) is the 3′primer.

TABLE A Heavy chain oligonucleotides used to amplify cDNA for Hco7 mice.Oligonucleotides 188 (SEQ ID NO: 1), 944 (SEQ ID NO: 2) and 948 (SEQ IDNO: 3) are 5′ primers and oligonucleotide 952 (SEQ ID NO: 4) is the 3′primer. OLIGO # 5′ TO 3′ SEQUENCE 188 TT ACC CCT GTG GCA AAA GCC GAA GTGCAG CTG GTG GAG TCT GG 944 TT ACC CCT GTG GCA AAA GCC CAG GTG CAG CTGGTG CAG TCT GG 948 TT ACC CCT GTG GCA AAA GCC CAG GTG CAG CTG GTG GAGTCT GG 952 GA TGG GCC CTT GGT GGA GGC

Table B. Kappa chain oligonucleotides used to amplify cDNA from Hco7mice, Hco12 mice, and Hco7/Co12 mice. Oligonucleotide 973 (SEQ ID NO:15)is the 3′ primer and the rest are 5′ primers.

TABLE B Kappa chain oligonucleotides used to amplify cDNA from Hco7mice, Hco12 mice, and Hco7/Co12 mice. Oligonucleotide 973 (SEQ ID NO:15) is the 3′ primer and the rest are 5′ primers. OLIGO # 5′ TO 3′SEQUENCE 189 CT GCC CAA CCA GCC ATG GCC GAA ATT GTG CTC ACC CAG TCT CC931 TC GCT GCC CAA CCA GCC ATG GCC GTC ATC TGG ATG ACC CAG TCT CC 932 TCGCT GCC CAA CCA GCC ATG GCC AAC ATC CAG ATG ACC CAG TCT CC 933 TC GCTGCC CAA CCA GCC ATG GCC GCC ATC CGG ATG ACC CAG TCT CC 934 TC GCT GCCCAA CCA GCC ATG GCC GCC ATC CAG TTG ACC CAG TCT CC 935 TC GCT GCC CAACCA GCC ATG GCC GAA ATA GTG ATG ACG CAG TCT CC 936 TC GCT GCC CAA CCAGCC ATG GCC GAT GTT GTG ATG ACA CAG TCT CC 937 TC GCT GCC CAA CCA GCCATG GCC GAA ATT GTG TTG ACG CAG TCT CC 955 TC GCT GCC CAA CCA GCC ATGGCC GAC ATC CAG ATG ATC CAG TCT CC 956 TC GCT GCC CAA CCA GCC ATG GCCGAT ATT GTG ATG ACC CAG ACT CC 973 CAG CAG GCA CAC AAC AGA GGC

Table C. Heavy chain oligonucleotides used to amplify cDNA for Hco12mice. Oligonucleotides 944, 945, 946, 947 and 948 (SEQ ID NOS:2, 16, 17,18, and 3 respectively) are 5′ primers and oligonucleotide 952 (SEQ IDNO:4) is the 3′ primer. The sequences of 944, 948 and 952 (SEQ ID NOS:2, 3, and 4 respectively) are shown in Table A.

TABLE C Heavy chain oligonucleotides used to amplify cDNA for Hco12mice. Oligonucleotides 944, 945, 946, 947 and 948 (SEQ ID NOS: 2, 16,17, 18, and 3 respectively) are 5′ primers and oligonucleotide 952 (SEQID NO: 4) is the 3′ primer. The sequences of 944, 948 and 952 (SEQ IDNOS: 2, 3, and 4 respectively) are shown in Table A. OLIGO # 5′ TO 3′SEQUENCE 945 TT ACC CCT GTG GCA AAA GCC GAG GTG CAG CTG TTG GAG TCT GG946 TT ACC CCT GTG GCA AAA GCC GAG GTG CAG CTG GTG CAG TCT GG 947 TT ACCCCT GTG GCA AAA GCC CAG GTG CAG CTA CAG CAG TGG GG

The dsDNA products of the PCR process were then subjected to asymmetricPCR using only 3′ primer to generate substantially only the anti-sensestrand of the target genes. Oligonucleotide 953 was used as the 3′primer for kappa chain asymmetric PCR (Table D) and oligonucleotide 952was used as the 3′ primer for heavy chain asymmetric PCR (Table A). Foreach spleen, two asymmetric reactions were run for the kappa chain PCRproducts to primer 189, 931, 932, 933, 934, 936, 955, and 956, (SEQ IDNOS: 5, 6, 7, 8, 9, 11, 13 and 14 respectively), four asymmetricreactions were run for the kappa chain PCR product to primer 935 (SEQ IDNO:10), and eight asymmetric reactions were run for the kappa chain PCRproduct to primer 937 (SEQ ID NO:12). The number of asymmetric reactionsused for each heavy chain PCR product was dependent on the mousegenotype. For Co7 mice, eight asymmetric reactions were run for each PCRproduct. For Co12 mice, eight asymmetric reactions were run for the PCRproduct from primer 944 (SEQ ID NO:2), and four asymmetric reactionswere run for the PCR products from the other primers. For Co7/Co12 mice,six asymmetric reactions were run for the PCR products from primers 944and 948 (SEQ ID NOS: 2 and 3), and three asymmetric reactions were runfor the PCR products from the other primers. Each reaction describedabove is 100 μL total volume with 200 pmol of 3′ primer, 2 μL of ds-DNAproduct, 0.5 μL Taq DNA Polymerase, 10 μL 2 mM dNTP's, 10 μL 10×Taq DNApolymerase buffer with MgCl₂, and H₂O to 100 μL. Heavy chain reactionswere amplified using the thermal profile described above, while kappachain reactions were amplified with the same thermal profile but 25cycles were used instead of 30 cycles.

TABLE D Oligonucleotide sequences used for asymmetric PCR of kappachains. OLIGO # 5′ TO 3′ SEQUENCE 953 GAC AGA TGG TGC AGC CAC AGT (SEQID NO:19)

EXAMPLE 4 Purification of ss-DNA by High Performance LiquidChromatography and Kinasing ss-DNA

The H chain ss-PCR products and the L chain ss-PCR products wereseparately pooled and ethanol precipitated by adding 2.5 volumes ethanoland 0.2 volumes 7.5 M ammonium acetate and incubating at −20° C. for atleast 30 min. The DNA was pelleted by centrifuging at 15,000 rpm for 15min at 2-8° C. The supernatant was carefully aspirated, and the tubeswere briefly spun a 2nd time. The last drop of supernatant was removedwith a pipet. The DNA was dried in vacuo for 10 min on medium heat. TheH chain products were dissolved in 210 μL water and the L chain productswere dissolved separately in 210 μL water. The ss-DNA was purified byhigh performance liquid chromatography (HPLC) using a Hewlett Packard1090 HPLC and a Gen-Pak™ FAX anion exchange column (Millipore Corp.,Milford, Mass.). The gradient used to purify the ss-DNA is shown inTable 1, and the oven temperature was at 60° C. Absorbance was monitoredat 260 nm. The ss-DNA eluted from the HPLC was collected in 0.5 minfractions. Fractions containing ss-DNA were pooled, ethanolprecipitated, pelleted and dried as described above. The dried DNApellets were resuspended in 200 μL sterile water.

TABLE 1 HPLC gradient for purification of ss-DNA Time (min) % A % B % CFlow (mL/min) 0 70 30 0 0.75 2 40 60 0 0.75 17 15 85 0 0.75 18 0 100 00.75 23 0 100 0 0.75 24 0 0 100 0.75 28 0 0 100 0.75 29 0 100 0 0.75 340 100 0 0.75 35 70 30 0 0.75 Buffer A is 25 mM Tris, 1 mM EDTA, pH 8.0Buffer B is 25 mM Tris, 1 mM EDTA, 1M NaCl, pH 8.0 Buffer C is 40 mmphosphoric acid

The ss-DNA was kinased on the 5′ end in preparation for mutagenesis(Example 7). 24 μL 10×kinase buffer (United States Biochemical,Cleveland, Ohio), 10.4 μL 10 mM adenosine-5′-triphosphate (BoehringerMannheim, Indianapolis, Ind.), and 2 μL polynucleotide kinase (30units/μL, United States Biochemical, Cleveland, Ohio) was added to eachsample, and the tubes were incubated at 37° C. for 1 hr. The reactionswere stopped by incubating the tubes at 70° C. for 10 min. The DNA waspurified with one extraction of equilibrated phenol (pH>8.0, UnitedStates Biochemical, Cleveland, Ohio)-chloroform-isoamyl alcohol(50:49:1) and one extraction with chloroform:isoamyl alcohol (49:1).After the extractions, the DNA was ethanol precipitated and pelleted asdescribed above. The DNA pellets were dried, then dissolved in 50 μLsterile water. The concentration was determined by measuring theabsorbance of an aliquot of the DNA at 260 nm using 33 μg/mL for anabsorbance of 1.0. Samples were stored at −20° C.

EXAMPLE 5 Construction of Antibody Phage Display Vector Having HumanAntibody Constant Region Sequences

The antibody phage display vector for cloning antibodies was derivedfrom an M13 vector supplied by Ixsys, designated 668-4. The vector 668-4contained the DNA sequences encoding the heavy and light chains of amouse monoclonal Fab fragment inserted into a vector described by Huse,WO 92/06024. The vector had a Lac promoter, a pelB signal sequence fusedto the 5′ side of the L chain variable region of the mouse antibody, theentire kappa chain of the mouse antibody, an alkaline phosphatase signalsequence at the 5′ end of the H chain variable region of the mouseantibody, the entire variable region and the first constant region ofthe H chain, and 5 codons of the hinge region of an IgG1 H chain. Adecapeptide sequence was at the 3′ end of the H chain hinge region andan amber stop codon separated the decapeptide sequence from thepseudo-gene VIII sequence. The amber stop allowed expression of H chainfusion proteins with the gene VIII protein in E. coli suppressor strainssuch as XL1 blue (Stratagene, San Diego, Calif.), but not innonsuppressor cell strains such as MK30 (Boehringer Mannheim,Indianapolis, Ind.) (see FIG. 1).

To make the first derivative cloning vector, deletions were made in thevariable regions of the H chain and the L chain by oligonucleotidedirected mutagenesis of a uracil template (Kunkel, Proc. Natl. Acad.Sci. USA 82:488 (1985); Kunkel, et al., Methods. Enzymol. 154:367(1987)). These mutations deleted the region of each chain from the 5′end of CDR1 to the 3′ end of CDR3, and the mutations added a DNAsequence where protein translation would stop (see FIG. 2 formutagenesis oligonucleotides). This prevented the expression of H or Lchain constant regions in clones without an insert, thereby allowingplaques to be screened for the presence of insert. The resulting cloningvector was called BS11.

Many changes were made to BS11 to generate the cloning vector used inthe present screening methods. The amber stop codon between the heavychain and the pseudo gene VIII sequence was removed so that every heavychain was expressed as a fusion protein with the gene VIII protein. Thisincreased the copy number of the antibodies on the phage relative toBS11. A HindIII restriction enzyme site in the sequence between the 3′end of the L chain and the 5′ end of the alkaline phosphatase signalsequence was deleted so antibodies could be subcloned into a pBR322derivative (Example 14). The interchain cysteine residues at thecarboxyl-terminus of the L and H chains were changed to serine residues.This increased the level of expression of the antibodies and the copynumber of the antibodies on the phage without affecting antibodystability. Nonessential DNA sequences on the 5′ side of the lac promoterand on the 3′ side of the pseudo gene VIII sequence were deleted toreduce the size of the M13 vector and the potential for rearrangement. Atranscriptional stop DNA sequence was added to the vector at the L chaincloning site to replace the translational stop so that phage with onlyheavy chain proteins on their surface, which might be nonspecifically inpanning, could not be made. Finally, DNA sequences for protein tags wereadded to different vectors to allow enrichment for polyvalent phage bymetal chelate chromatography (polyhistidine sequence) or by affinitypurification using a decapeptide tag and a magnetic latex having animmobilized antibody that binds the decapeptide tag. BS45 had apolyhistidine sequence between the end of the heavy chain constantregion and the pseudo-gene VIII sequence, and a decapeptide sequence atthe 3′ end of the kappa chain constant region.

The mouse heavy and kappa constant region sequences were deleted fromBS45 by oligonucleotide directed mutagenesis. Oligonucleotide 864 wasused to delete the mouse kappa chain and oligonucleotide 862 was used todelete the mouse heavy chain.

Oligonucleotide 864 (SEQ ID NO:20)

5′ ATC TGG CAC ATC ATA TGG ATA AGT TTC GTG TAC AAA ATG CCA GAC CTA GAGGAA TTT TAT TTC CAG CTT GGT CCC

Oligonucleotide 862 (SEQ ID NO:21)

5′ GTG ATG GTG ATG GTG ATG GAT CGG AGT ACC AGG TTA TCG AGC CCT CGA TATTGA GGA GAC GGT GAC TGA

Deletion of both constant region sequences was determined by amplifyingthe DNA sequence containing both constant regions by PCR usingoligonucleotides 5 and 197, followed by sizing the PCR products on DNAagarose gel. The PCR was accomplished as described in Example 3 for thedouble-stranded DNA, except 1 μL of phage was template instead of cDNA.Phage with the desired deletion had a shorter PCR product than onedeletion or no deletion. Uracil template was made from one phage stockhaving both deletions, as described in Example 6. This template, BS50,was used to insert the human constant region sequences for the kappachain and IgG1.

Primer 5 (SEQ ID NO:22)

5′ GCA ACT GTT GGG AAG GG

Primer 197 (SEQ ID NO:23)

5′ TC GCT GCC CAA CCA GCC ATG

The human constant region DNA sequences were amplified from human spleencDNA (Clontech, Palo Alto, Calif.). Oligonucleotides 869 and 870 wereused to amplify the kappa constant region sequence, and oligonucleotides867 and 876 were used to amplify the IgG1 constant region sequence andthe codons for 6 amino acids of the hinge region (Kabat et al.,Sequences of Proteins of Immunological Interest, 1991).

5′ PCR primer (869)—GGG ACC AAG CTG GAA ATA AAA CGG GCT GTG GCT GCA CCATCT GTC T (SEQ ID NO:24)

3′ PCR primer (870)—ATC TGG CAC ATC ATA TGG ATA AGA CTC TCC CCT GTT GAAGCT CTT (SEQ ID NO:25)

5′ PCR primer (867)—TCA GTC ACC GTC TCC TCA GCC TCC ACC AAG GGC CCA TC(SEQ ID NO:26)

3′ PCR primer (876)—GTG ATG GTG ATG GTG ATG AGA TTT GGG CTC TGC TTT CTTGTC C (SEQ ID NO:27)

PCR (1-50 μL reaction for each chain) was performed using Expandhigh-fidelity PCR system (Roche Molecular Biochemicals, Indianapolis,Ind.). Each 50 μL reaction contained 50 pmol of 5′ primer, 50 pmol of 3′primer, 0.35 units of Expand DNA polymerase, 5 μL 2 mM dNTP's, 5 μL10×Expand reaction buffer, 1 μL cDNA as template, and water to 50 μL.The reaction was carried out in a Perkin-Elmer thermal cycler (Model9600) using the following thermal profile for the kappa chain: one cycleof denaturation at 94° C. (1 min); ten cycles of denaturation (15 sec,94° C.), annealing (30 sec, 55° C.), elongation (60 sec, 72° C.);fifteen cycles of denaturation (15 sec, 94° C.), annealing (30 sec, 55°C.), elongation (80 sec plus 20 sec for each additional cycle, 72° C.);elongation (6 min, 72° C.); soak (4° C., indefinitely). The thermalprofile used for the heavy chain reaction had twenty cycles instead offifteen in the second part of the thermal profile.

The dsDNA products of the PCR process were then subjected to asymmetricPCR using only 3′ primer to generate substantially only the anti-sensestrand of the human constant region genes, as described in Example 3.Five reactions were done for the kappa chain and ten reactions were donefor the heavy chain (100 μL per reaction). The thermal profile for bothconstant region genes is the same as that described in Example 3,including the heavy chain asymmetric PCR was done with 30 cycles and thekappa chain asymmetric PCR was done with 25 cycles. The single strandedDNA was purified by HPLC as described in Example 4. The HPLC purifiedkappa chain DNA was dissolved in 55 μL of water and the HPLC purifiedheavy chain was dissolved in 100 μL of water. The DNA was quantified byabsorbance at 260 nm, as described in Example 4, then the DNA waskinased as described in Example 4 except added 6 μL 10×kinase buffer,2.6 μL 10 mM ATP, and 0.5 μL of polynucleotide kinase to 50 μL of kappachain DNA. Twice those volumes of kinase reagents were added to 100 μLof heavy chain DNA.

The kinased DNA was used to mutate BS50 without purifying the DNA byextractions. The mutagenesis was performed on a 2 μg scale by mixing thefollowing in a 0.2 mL PCR reaction tube: 8 μl of (250 ng/μl) BS50 uraciltemplate, 8 μl of 10×annealing buffer (200 mM Tris pH 7.0, 20 mM MgCl₂,500 mM NaCl), 2.85 μl of kinased single-stranded heavy chain insert (94ng/μl), 6.6 μl of kinased single-stranded kappa chain insert (43.5ng/μl), and sterile water to 80 μl. DNA was annealed in a GeneAmp® 9600thermal cycler using the following thermal profile: 20 sec at 94° C.,85° C. for 60 sec, 85° C. to 55° C. ramp over 30 min, hold 55° C. for 15min. The DNA was transferred to ice after the program finished. Theextension/ligation was carried out by adding 8 μl of 10×synthesis buffer(5 mM each dNTP, 10 mM ATP, 100 mM Tris pH 7.4, 50 mM MgCl₂, 20 mM DTT),8 μl T4 DNA ligase (1 U/μl, Roche Molecular Biochemicals, Indianapolis,Ind.), 8 μl diluted T7 DNA polymerase (1 U/μl, New England BioLabs,Beverly, Mass.) and incubating at 37° C. for 30 min. The reaction wasstopped with 296 μl of mutagenesis stop buffer (10 mM Tris pH 8.0, 10 mMEDTA). The mutagenesis DNA was extracted once with equilibrated phenol(pH>8):chloroform:isoamyl alcohol (50:49:1), once withchloroform:isoamyl alcohol (49:1), and the DNA was ethanol precipitatedat −20° C. for at least 30 min. The DNA was pelleted and the supernatantcarefully removed as described above. The sample was briefly spun againand all traces of ethanol removed with a pipetman. The pellet was driedin vacuo. The DNA was resuspended in 4 μl of sterile water. 1 μlmutagenesis DNA was (500 ng) was transferred into 40 μl electrocompetentE. coli DH12S (Gibco/BRL, Gaithersburg, Md.) using the electroporationconditions in Example 8. The transformed cells were mixed with 1.0 mL2×YT broth (Sambrook, et al., supra) and transferred to a 15 mL sterileculture tube. Aliquots (10 μL of 10⁻³ and 10⁻⁴ dilutions) of thetransformed cells were plated on 100 mm LB agar plates as described inExample 11. After 6 hr of growth at 37° C., 20 individual plaques werepicked from a plate into 2.75 mL 2×YT and 0.25 ml overnight XL1 bluecells. The cultures were grown at 37° C., 300 rpm overnight to amplifythe phage from the individual plaques. The phage samples were analyzedfor insertion of both constant regions by PCR using oligonucleotides 197and 5 (see above in BS50 analysis), followed by sizing of the PCRproducts by agarose gel electrophoresis. The sequence of two cloneshaving what appeared to be two inserts by agarose gel electrophoresiswas verified at MacConnell Research (San Diego, Calif.) by the dideoxychain termination method using a Sequatherm sequencing kit (EpicenterTechnologies, Madison, Wis.) and a LI-COR 4000L automated sequencer(LI-COR, Lincoln, Nev.). Oligonucleotide primers 885 and 5, that bind onthe 3′ side of the kappa chain and heavy chain respectively, were used.Both clones had the correct sequence. The uracil template having humanconstant region sequences, called BS46, was prepared as described inExample 6.

Primer 885

5′ TAA GAG CGG TAA GAG TGC CAG (SEQ ID NO:28)

EXAMPLE 6 Preparation of Uracil Templates Used in Generation of SpleenAntibody Phage Libraries

1 mL of E. coli CJ236 (BioRAD, Hercules, Calif.) overnight culture and10 μL of a 1/100 dilution of vector phage stock was added to 50 ml 2×YTin a 250 mL baffled shake flask. The culture was grown at 37° C. for 6hr. Approximately 40 mL of the culture was centrifuged at 12,000 rpm for15 minutes at 4° C. The supernatant (30 mL) was transferred to a freshcentrifuge tube and incubated at room temperature for 15 minutes afterthe addition of 15 μl of 10 mg/ml RnaseA (Boehringer Mannheim,Indianapolis, Ind.). The phage were precipitated by the addition of 7.5ml of 20% polyethylene glycol 8000 (Fisher Scientific, Pittsburgh,Pa.)/3.5M ammonium acetate (Sigma Chemical Co., St. Louis, Mo.) andincubation on ice for 30 min. The sample was centrifuged at 12,000 rpmfor 15 min at 2-8° C. The supernatant was carefully discarded, and thetube was briefly spun to remove all traces of supernatant. The pelletwas resuspended in 400 μl of high salt buffer (300 mM NaCl, 100 mM TrispH 8.0, 1 mM EDTA), and transferred to a 1.5 mL tube. The phage stockwas extracted repeatedly with an equal volume of equilibratedphenol:chloroform:isoamyl alcohol (50:49:1) until no trace of a whiteinterface was visible, and then extracted with an equal volume ofchloroform:isoamyl alcohol (49:1). The DNA was precipitated with 2.5volumes of ethanol and ⅕ volume 7.5 M ammonium acetate and incubated 30min at −20° C. The DNA was centrifuged at 14,000 rpm for 10 min at 4°C., the pellet washed once with cold 70% ethanol, and dried in vacuo.The uracil template DNA was dissolved in 100 μl sterile water and theconcentration determined by A₂₆₀ using an absorbance of 1.0 for aconcentration of 40 μg/ml. The template was diluted to 250 ng/μl withsterile water, aliquoted, and stored at −20° C.

EXAMPLE 7 Mutagenesis of Uracil Template with ss-DNA and ElectroporationInto E. coli to Generate Antibody Phage Libraries

Antibody phage-display libraries were generated by simultaneouslyintroducing single-stranded heavy and light chain genes onto aphage-display vector uracil template. A typical mutagenesis wasperformed on a 2 μg scale by mixing the following in a 0.2 mL PCRreaction tube: 8 μl of (250 ng/μl) BS46 uracil template (examples 5 and6), 8 μl of 10×annealing buffer (200 mM Tris pH 7.0, 20 mM MgCl₂, 500 mMNaCl), 3.33 μl of kinased single-stranded heavy chain insert (100ng/μl), 3.1 μl of kinased single-stranded light chain insert (100ng/ml), and sterile water to 80 μl. DNA was annealed in a GeneAmp® 9600thermal cycler using the following thermal profile: 20 sec at 94° C.,85° C. for 60 sec, 85° C. to 55° C. ramp over 30 min, hold at 55° C. for15 min. The DNA was transferred to ice after the program finished. Theextension/ligation was carried out by adding 8 μl of 10×synthesis buffer(5 mM each dNTP, 10 mM ATP, 100 mM Tris pH 7.4, 50 mM MgCl₂, 20 mM DTT),8 μl T4 DNA ligase (1 U/μl), 8 μl diluted T7 DNA polymerase (1 U/μl) andincubating at 37° C. for 30 min. The reaction was stopped with 300 μl ofmutagenesis stop buffer (10 mM Tris pH 8.0, 10 mM EDTA). The mutagenesisDNA was extracted once with equilibrated phenol(pH>8):chloroform:isoamyl alcohol (50:49:1), once withchloroform:isoamyl alcohol (49:1), and the DNA was ethanol precipitatedat −20° C. for at least 30 min. The DNA was pelleted and the supernatantcarefully removed as described above. The sample was briefly spun againand all traces of ethanol removed with a pipetman. The pellet was driedin vacuo. The DNA was resuspended in 4 μl of sterile water. 1 μlmutagenesis DNA was (500 ng) was transferred into 40 μl electrocompetentE. coli DH12S (Gibco/BRL, Gaithersburg, Md.) using the electroporationconditions in Example 8. The transformed cells were mixed with 0.4 mL2×YT broth (Sambrook, et al., supra) and 0.6 mL overnight XL1 Bluecells, and transferred to 15 mL sterile culture tubes. The first roundantibody phage samples were generated by plating the electroporatedsamples on 150 mm LB plates as described in Example 11. The plates wereincubated at 37° C. for 4 hr, then 20° C. overnight. The first roundantibody phage was eluted from the 150 mm plates by pipeting 10 mL 2YTmedia onto the lawn and gently shaking the plate at room temperature for20 min. The phage were transferred to 15 mL disposable sterilecentrifuge tubes with plug seal cap and the debris from the LB plate waspelleted by centrifuging for 15 min at 3500 rpm. The 1^(st) roundantibody phage was then transferred to a new tube.

The efficiency of the electroporation was measured by plating 10 μl of10⁻³ and 10⁻⁴ dilutions of the cultures on LB agar plates (see Example11). These plates were incubated overnight at 37° C. The efficiency wasdetermined by multiplying the number of plaques on the 10⁻³ dilutionplate by 10⁵ or multiplying the number of plaques on the 10⁻⁴ dilutionplate by 10⁶.

EXAMPLE 8 Transformation of E. coli by Electroporation

The electrocompetent E. coli cells were thawed on ice. DNA was mixedwith 20-40 μL electrocompetant cells by gently pipetting the cells upand down 2-3 times, being careful not to introduce air-bubble. The cellswere transferred to a Gene Pulser cuvette (0.2 cm gap, BioRAD, Hercules,Calif.) that had been cooled on ice, again being careful not tointroduce an air-bubble in the transfer. The cuvette was placed in theE. coli Pulser (BioRAD, Hercules, Calif.) and electroporated with thevoltage set at 1.88 kV according to the manufacturer's recommendation.The transformed sample was immediately diluted to 1 ml with 2×YT brothor 1 ml of a mixture of 400 μL 2×YT/600 μL overnight XL1 Blue cells andprocessed as procedures dictate.

EXAMPLE 9 Preparation of Biotinylated Interleukin 8 (IL8)

IL8 was dialyzed against a minimum of 100 volumes of 20 mM borate, 150mM NaCl, pH 8 (BBS) at 2-8° C. for at least 4 hr. The buffer was changedat least once prior to biotinylation. IL8 was reacted with biotin-XX-NHSester (Molecular Probes, Eugene, Oreg., stock solution at 40 mM indimethylformamide) at a final concentration of 1 mM for 1 hr at roomtemperature. After 1 hr, the IL8 was extensively dialyzed into BBS toremove unreacted small molecules.

EXAMPLE 10 Preparation of Avidin Magnetic Latex

The magnetic latex (superparamagnetic microparticles, 0.96 μm, Estapor,10% solids, Bangs Laboratories, Carmel, Ind.) was thoroughly resuspendedand 2 ml aliquoted into a 15 ml conical tube. The magnetic latex wassuspended in 12 ml distilled water and separated from the solution for10 min using a magnet. While still in the magnet, the liquid wascarefully removed with a 10 mL sterile pipet. This washing process wasrepeated an additional three times. After the final wash, the latex wasresuspended in 2 ml of distilled water. In a separate 50 ml conicaltube, 10 mg of avidin-HS (NeutrAvidin, Pierce, Rockford, Ill.) wasdissolved in 18 ml of 40 mM Tris, 0.15 M sodium chloride, pH 7.5 (TBS).While vortexing, the 2 ml of washed magnetic latex was added to thediluted avidin-HS and the mixture vortexed an additional 30 seconds.This mixture was incubated at 45° C. for 2 hr, shaking every 30 minutes.The avidin magnetic latex was separated from the solution using a magnetand washed three times with 20 ml BBS as described above. After thefinal wash, the latex was resuspended in 10 ml BBS and stored at 4° C.

Immediately prior to use, the avidin magnetic latex was equilibrated inpanning buffer (40 mM TRIS, 1.50 mM NaCl, 20 mg/mL BSA, 0.1% Tween 20(Fisher Scientific, Pittsburgh, Pa.), pH 7.5). The avidin magnetic latexneeded for a panning experiment (200 μl/sample) was added to a sterile15 ml centrifuge tube and brought to 10 ml with panning buffer. The tubewas placed on the magnet for 10 min to separate the latex. The solutionwas carefully removed with a 10 mL sterile pipet as described above. Themagnetic latex was resuspended in 10 mL of panning buffer to begin thesecond wash. The magnetic latex was washed a total of 3 times withpanning buffer. After the final wash, the latex was resuspended inpanning buffer to the initial aliquot volume.

EXAMPLE 11 Plating M13 Phage or Cells Transformed with AntibodyPhage-display Vector Mutagenesis Reaction

The phage samples were added to 200 μL of an overnight culture of E.coli XL1-Blue when plating on 100 mm LB agar plates or to 600 μL ofovernight cells when plating on 150 mm plates in sterile 15 ml culturetubes. The electroporated phage samples were in 1 mL 2×YT/overnight XL1cells, as described in Example 8, prior to plating on 150 mm plates.After adding LB top agar (3 mL for 100 mm plates or 9 mL for 150 mmplates, top agar stored at 55° C., Appendix A1, Molecular Cloning, ALaboratory Manual, (1989) Sambrook. J), the mixture was evenlydistributed on an LB agar plate that had been pre-warmed (37° C.-55° C.)to remove any excess moisture on the agar surface. The plates werecooled at room temperature until the top agar solidified. The plateswere inverted and incubated at 37° C. as indicated.

EXAMPLE 12 Develop Nitrocellulose Filters with Alkaline Phosphatase (AP)Conjugates

After overnight incubation of the nitrocellulose filters on LB agarplates, the filters were carefully removed from the plates with membraneforceps and incubated for 2 hr in block (1% bovine serum albumin (from30% BSA, Bayer, Kankakee, Ill.), 10 mM Tris, 150 mM NaCl, 1 mM MgCl₂,0.1 mM ZnCl₂, 0.1% polyvinyl alcohol (80% hydrolyzed, Aldrich ChemicalCo., Milwaukee, Wis.), pH 8.0).

After 2 hr, the filters were incubated with goat anti-human kappa AP(Southern Biotechnology Associates, Inc, Birmingham, Ala.) for 2-4 hr.The AP conjugate was diluted into block at a final concentration of 1μg/mL. Filters were washed 3 times with 40 mM TRIS, 150 mM NaCl, 0.05%Tween 20, pH 7.5 (TBST) (Fisher Chemical, Pittsburgh, Pa.) for 5 mineach. After the final wash, the filters were developed in a solutioncontaining 0.2 M 2-amino-2-methyl-1-propanol (JBL Scientific, San LuisObispo, Calif.), 0.5 M TRIS, 0.33 mg/mL nitro blue tetrazolium (FisherScientific, Pittsburgh, Pa.) and 0.166 mg/mL5-bromo-4-chloro-3-indolyl-phosphate, p-toluidine salt.

EXAMPLE 13 Enrichment of Polyclonal Phage to Human Interleukin-8 Using aDecapeptide Tag on the Kappa Chain

The first round antibody phage was prepared as described in Example 7using BS46 uracil template, which has a decapeptide tag for polyvalentenrichment fused to the kappa chain. Fourteen electroporations ofmutagenesis DNA were done from 7 different spleens (2 electroporationsfrom each spleen) yielding 14 different phage samples. Prior tofunctional panning, the antibody phage samples were enriched forpolyvalent display using the decapeptide tag on the kappa chain and the7F11 magnetic latex. Binding studies had previously shown that thedecapeptide could be eluted from the monoclonal antibody 7F11 (seeExample 17) at a relatively mild pH of 10.5-11. The 7F11 magnetic latex(2.9 mL) was equilibrated with panning buffer as described above for theavidin magnetic latex (Example 10). Each first round phage stock (1 mL)was aliquoted into a 15 mL tube. The 7F11 magnetic latex (200 μL perphage sample) was incubated with phage for 10 min at room temperature.After 10 min, 9 mL of panning buffer was added, and the magnetic latexwas separated from unbound phage by placing the tubes in a magnet for 10min. After 10 min in the magnet, the unbound phage was carefully removedwith a 10 mL sterile pipet. The magnetic latex was then resuspended in 1mL panning buffer and transferred to 1.5 mL tubes. The magnetic latexwas separated from unbound phage by placing the tubes in a smallermagnet for 5 min, then the supernatant was carefully removed with asterile pipet. The latexes were washed with 1 additional 1 mL panningbuffer wash. Each latex was resuspended in 1 mL elution buffer (20 mM3-(cyclohexylamino)propanesulfonic acid (United States Biochemical,Cleveland, Ohio), 150 mM NaCl, 20 mg/mL BSA, pH 10.5) and incubated atroom temperature for 10 min. After 10 min, tubes were placed in thesmall magnet again for 5 min and the eluted phage was transferred to anew 1.5 mL tube. The phage samples were again placed in the magnet for 5min to remove the last bit of latex that was transferred. Eluted phagewas carefully removed into a new tube and 25 μL 3 M Tris, pH 6.8 wasadded to neutralize the phage. Panning with IL8-biotin was set up foreach sample by mixing 900 μL 7F11/decapeptide enriched phage, 100 μLpanning buffer, and 10 μL 10⁻⁷ M IL8-biotin and incubating overnight at2-8° C.

The antibody phage samples were panned with avidin magnetic latex. Theequilibrated avidin magnetic latex (see Example 11), 200 μL latex persample, was incubated with the phage for 10 min at room temperature.After 10 min, approximately 9 mL of panning buffer was added to eachphage sample, and the magnetic latex was washed as described above forthe 7F11 magnetic latex. A total of one 9 mL and three 1 mL panningbuffer washes were done. After the last wash, each latex was resuspendedin 200 μL 2×YT, then the entire latex of each sample was plated on 150mm LB plates to generate the 2nd round antibody phage. The 150 mm plateswere incubated at 37° C. for 4 hr, then overnight at 20° C.

The resulting 2^(nd) round antibody phage samples were set up for thesecond round of functional panning in separate 15 mL disposable sterilecentrifuge tubes with plug seal cap by mixing 900 μL panning buffer, 100μL 2^(nd) round antibody phage, and 10 μL 10⁻⁷M interleukin-8-biotin.After overnight incubation at 2-8° C., the phage samples were pannedwith avidin magnetic latex as described above. Aliquots of one samplefrom each spleen were plated on 100 mm LB agar plates to determine thepercentage of kappa positives (Example 12). The percentage of kappapositives for the 2nd round of panning was between 83-92% for 13samples. One sample was discarded because it was 63% kappa positive.

The remaining thirteen samples were set up for a third round offunctional panning as described above using 950 μL panning buffer, 50 μL3^(rd) round antibody phage, and 10 μL 10⁻⁶M interleukin-8-biotin. Afterincubation for 1.5 hours at 2-8° C., the phage samples were panned withavidin magnetic latex, and nitrocellulose filters were placed on eachphage sample, as described above. The percentage of kappa positives forthe 4th round antibody phage samples was estimated to be greater than80%.

The 4th round antibody phage samples were titered by plating 50 μL 10⁻⁸dilutions on 100 mm LB plates. After 6 hr at 37° C., the number ofplaques on each plate were counted, and the titers were calculated bymultipying the number of plaques by 2×10⁹. A pool of 13—4th round phagewas made by mixing an equal number of phage from each phage stock sothat high titer phage stocks would not bias the pool. The pooledantibody phage was set up in duplicate for a 4^(th) round of functionalpanning as described above using 950 μL panning buffer, 50 μL 4th roundpooled-antibody phage. One sample (foreground) received 10 μL 10⁻⁶Minterleukin-8-biotin and the other sample (background) did not receiveinterleukin-8-biotin and served as a blank to monitor non-specificbinding of phage to the magnetic latex. After incubation for 1.5 hoursat 2-8° C., the phage samples were panned with avidin magnetic latex asdescribed above. The next day, the 5^(th) round antibody phage waseluted and the number of plaques was counted on the foreground andbackground plates. The foreground:background ratio was 58:1.

The 5^(th) round antibody phage was set up in triplicate as describedabove using 950 μL panning buffer, 50 μL 5th round antibody phage persample with the experimental (foreground) tubes receiving 10 μL 10⁻⁷Minterleukin-8-biotin or 10 μL 10⁻⁸M interleukin-8-biotin, respectively.The third tube did not receive any interleukin-8-biotin. This round ofpanning or affinity selection preferentially selects for antibodies of>10⁹ affinity and >10¹⁰ affinity by including the interleukin-8-biotinat a final concentration of 10⁻⁹ M and 10⁻¹⁰ M, respectively. Aftergreater than 24 hours at 2-8° C., the phage samples were panned withavidin magnetic latex and processed as described above. The 6^(th) roundantibody phage sample 10⁻⁹ M cut had a foreground:background ratio1018:1 and the 10⁻¹⁰M cut had a foreground:background ratio 225:1.

An additional round of panning was done on the 6^(th) round 10⁻¹⁰ M cutantibody phage to increase the number of antibodies with affinity of10¹⁰. The 6^(th) round phage were set up as described above using 975 μLpanning buffer, 25 μL 6th round antibody phage per sample with theexperimental (foreground) tube receiving 10 μL 10⁻⁸Minterleukin-8-biotin. The blank did not receive anyinterleukin-8-biotin. After overnight incubation at 2-8° C., the phagesamples were panned with avidin magnetic latex and processed asdescribed above. The 7^(th) round antibody phage sample 10⁻¹⁰ M cut hada foreground:background ratio 276:1. The antibody phage populations weresubcloned into the expression vector and electroporated as described inExample 15.

EXAMPLE 14 Construction of the pBR Expression Vector

An expression vector and a process for the subcloning of monoclonal andpolyclonal antibody genes from a phage-display vector has been developedthat is efficient, does not substantially bias the polyclonalpopulation, and can select for vector containing an insert capable ofrestoring antibiotic resistance. The vector is a modified pBR322plasmid, designated pBRncoH3, that contains an arabinose promoter,ampicillin resistance (beta-lactamase) gene, a partial tetracyclineresistance gene, a pelB (pectate lyase) signal sequence, and NcoI andHindIII restriction sites. (FIG. 3). The pBRncoH3 vector can also beused to clone proteins other than Fabs with a signal sequence. A secondvector, pBRnsiH3, has been developed for cloning proteins with orwithout signal sequences, identical to the vector described above exceptthat the pelB signal sequence is deleted and the NcoI restriction sitehas been replaced with an NsiI site.

The araC regulatory gene (including the araBAD promoter) was amplifiedfrom E. coli K-12 strain NL31-001 (a gift from Dr. Nancy Lee at UCSB) byPCR (Example 3) using Taq DNA polymerase (Boehringer Mannheim,Indianapolis, Ind.) with primers A and B (Table 3). Primers A and Bcontain 20 base-pairs of the BS39 vector sequence at their 5′-endscomplementary to the 5′ side of the lac promoter and the 5′ side of thepelB signal sequence, respectively. Primer A includes an EcoRIrestriction site at its 5′-end used later for ligating the ara insertinto the pBR vector. The araCparaBAD PCR product was verified by agarosegel electrophoresis and used as template for an asymmetric PCR reactionwith primer ‘B’ in order to generate the anti-sense strand of theinsert. The single-stranded product was run on agarose gelelectrophoresis, excised, purified with GeneClean (Bio101, San Diego,Calif.), and resuspended in water as per manufacturers recommendations.The insert was kinased with T4 polynucleotide kinase for 45 min at 37°C. The T4 polynucleotide kinase was heat inactivated at 70° C. for 10min and the insert extracted with an equal volume of phenol/chloroform,followed by chloroform. The DNA was precipitated with ethanol at −20° C.for 30 min. The DNA was pelleted by centrifugation at 14 krpm for 15 minat 4° C., washed with ice-cold 70% ethanol, and dried in vacuo.

The insert was resuspended in water and the concentration determined byA₂₆₀ using an absorbance of 1.0 for a concentration of 40 μg/ml. Theinsert was cloned into the phage-display vector BS39 for sequenceverification and to introduce the pelB signal sequence in frame with thearabinose promoter (the pelB signal sequence also contains a NcoIrestriction site at its 3′-end used later for ligating the ara insertinto the pBR vector). The cloning was accomplished by mixing 250 ng ofBS39 uracil template (Example 5), 150 ng of kinased araCpBAD insert, and1.0 μl of 10×annealing buffer in a final volume of 10 μl. The sample washeated to 70▭ C. for 2 min and cooled over 20 min to room temperature toallow the insert and vector to anneal. The insert and vector wereligated together by adding 1 μl of 10×synthesis buffer, 1 μl T4 DNAligase (1U/μl), 1 μl T7 DNA polymerase (1 U/μl) and incubating at 37° C.for 30 min. The reaction was stopped with 90 μl of stop buffer (10 mMTris pH 8.0, 10 mM EDTA) and 1 μl electroporated (Example 8) intoelectrocompetent E. coli strain, DH10B, (Life Technologies,Gaithersburg, Md.).

The transformed cells were diluted to 1.0 ml with 2×YT broth and 1 10μl, 100 μl plated as described in Example 12. Following incubationovernight at 37° C., individual plaques were picked, amplified by PCRwith primers A and B, and checked for full-length insert by agarose gelelectrophoresis. Clones with full-length insert were sequenced withprimers D, E, F, G (Table 3) and checked against the literature. Aninsert with the correct DNA sequence was amplified by PCR (Example 3)from BS39 with primers A and C (FIG. 4A) and the products run on agarosegel electrophoresis.

Full-length products were excised from the gel and purified as describedpreviously and prepared for cloning by digestion with EcoRI and NcoI. ApBR lac-based expression vector that expressed a murine Fab was preparedto receive this insert by EcoRI and NcoI digestion. This digestionexcised the lac promoter and the entire coding sequence up to the 5′-endof the heavy chain (C_(H)1) constant region (FIG. 4A).

The insert and vector were mixed (2:1 molar ratio) together with 1 μl 10mM ATP, 1 μl (1U/μl) T4 DNA ligase, 1 μl 10×ligase buffer in a finalvolume of 10 μl and ligated overnight at 15° C. The ligation reactionwas diluted to 20 μl, and 1 μl electroporated into electrocompetent E.coli strain, DH10B (Example 8), plated on LB tetracycline (10 μg/ml)plates and grown overnight at 37° C.

Clones were picked and grown overnight in 3 ml LB broth supplementedwith tetracycline at 20 μg/ml. These clones were tested for the correctinsert by PCR amplification (Example 3) with primers A and C, using 1 μlof overnight culture as template. Agarose gel electrophoresis of the PCRreactions demonstrated that all clones had the araCparaB insert. Thevector (plasmid) was purified from each culture by Wizard miniprepcolumns (Promega, Madison, Wis.) following manufacturersrecommendations. The new vector contained the araC gene, the araBpromoter, the pelB signal sequence, and essentially the entire C_(H)1region of the heavy chain (FIG. 4B).

The vector was tested for expression by re-introducing the region of theFab that was removed by EcoRI and NcoI digestion. The region wasamplified by PCR, (Example 3) from a plasmid (20 ng) expressing 14F8with primers H and I (Table 3). The primers, in addition to havingsequence specific to 14F8, contain 20 base-pairs of vector sequence attheir 5′-end corresponding to the 3′-end of the pelB signal sequence andthe 5′-end of the C_(H)1 region for cloning purposes. The PCR productswere run on agarose gel electrophoresis and full-length products excisedfrom the gel and purified as described previously.

The vector was linearized with NcoI and together with the insert,prepared for cloning through the 3′→5′ exonuclease activity of T4 DNApolymerase. The insert and NcoI digested vector were prepared for T4exonuclease digestion by aliquoting 1.0 μg of each in separate tubes,adding 1.0 μl of 10×restriction endonuclease Buffer A (BoehringerMannheim, Indianapolis, Ind.) and bringing the volume to 9.0 μl withwater. The samples were digested for 5 min at 30° C. with 1 μl (1U/μl)of T4 DNA polymerase. The T4 DNA polymerase was heat inactivated byincubation at 70° C. for 15 min. The samples were cooled, briefly spun,and the digested insert (35 ng) and vector (100 ng) mixed together andthe volume brought to 10 μl with 1 mM MgCl₂. The sample was heated to70° C. for 2 min and cooled over 20 min to room temperature to allow thecomplementary 5′ single-stranded overhangs of the insert and vectorresulting from the exonuclease digestion to anneal together (FIG. 5).The annealed DNA (1.5 μl) was electroporated (Example 8) into 30 μl ofelectrocompetent E. coli strain DH10B.

The transformed cells were diluted to 1.0 ml with 2×YT broth and 1 μl,10 μl, and 100 μl plated on LB agar plates supplemented withtetracycline (10 μg/ml) and grown overnight at 37° C. The following day,two clones were picked and grown overnight in 2×YT (10 μg/mltetracycline) at 37° C. To test protein expression driven from the arapromoter, these cultures were diluted 1/50 in 2×YT(tet) and grown toOD₆₀₀=1.0 at which point they were each split into two cultures, one ofwhich was induced by the addition of arabinose to a final concentrationof 0.2% (W/V). The cultures were grown overnight at room temperature,and assayed for Fab production by ELISA. Both of the induced cultureswere producing approximately 20 μg/ml Fab. There was no detectable Fabin the uninduced cultures.

Initial efforts to clone polyclonal populations of Fab were hindered bybackgrounds of undigested vector ranging from 3-13%. This undigestedvector resulted in loss of Fab expressing clones due to the selectiveadvantage non-expressing clones have over Fab expressing clones. Avariety of means were tried to eliminate undigested vector from thevector preparations with only partial success; examples including:digesting the vector overnight 37° C. with NcoI, extracting, andredigesting the preparation a second time; including spermidine in theNcoI digest; including single-stranded binding protein (United StatesBiochemical, Cleveland, Ohio) in the NcoI digest; preparative gelelectrophoresis. It was then noted that there is a HindIII restrictionsite in pBR, 19 base-pairs from the 5′-end of the tetracycline promoter.A vector missing these 19 base-pairs is incapable of supporting growthin the presence of tetracycline, eliminating background due toundigested vector.

The ara-based expression vector was modified to make it tetracyclinesensitive in the absence of insert. This was done by digesting thepBRnco vector with NcoI and HindIII (Boehringer Mannheim, Indianapolis,Ind.), which removed the entire antibody gene cassette and a portion ofthe tet promoter (FIG. 4B). The region excised by NcoI/HindIII digestionwas replaced with a stuffer fragment of unrelated DNA by ligation asdescribed above. The ligation reaction was diluted to 20 μl, and 1 μlelectroporated (Example 8) into electrocompetent E. coli strain DH10B,plated on LB ampicillin (100 μg/ml) and incubated at 37° C.

After overnight incubation, transformants were picked and grownovernight in LB broth supplemented with ampicillin (100 μg/ml). Thevector (plasmid) was purified from each culture by Wizard miniprepcolumns following manufacturers recommendations. This modified vector,pBRncoH3, is tet sensitive, but still retains ampicillin resistance forgrowing preparations of the vector.

The antibody gene inserts were amplified by PCR with primers I and J(Table 3) as described in Example 3; primer J containing the 19base-pairs of the tet promoter removed by HindIII digestion, in additionto 20 base-pairs of vector sequence 3′ to the HindIII site forannealing. This modified vector was digested with NcoI/HindIII and,together with the insert, exonuclease digested and annealed as describedpreviously. The tet resistance is restored only in clones that containan insert capable of completing the tet promoter. The annealedFab/vector (1 μl) was transformed (Example 8) into 30 μl ofelectrocompetent E. coli strain, DH10B.

The transformed cells were diluted to 1.0 ml with 2×YT broth and 10 μlof 10⁻² and 10⁻³ dilutions plated on LB agar plates supplemented withtetracycline at 10 μg/ml to determine the size of the subclonedpolyclonal population. This plating also provides and opportunity topick individual clones from the polyclonal if necessary. The remainingcells were incubated at 37° C. for 1 hr and then diluted 1/100 into 30ml 2×YT supplemented with 1% glycerol and 20 μg/ml tetracycline andgrown overnight at 37° C. The overnight culture was diluted 1/100 intothe same media and grown 8 hr at which time glycerol freezer stocks weremade for long term storage at −80° C.

The new vector eliminates growth bias of clones containing vector only,as compared to clones with insert. This, together with the arabinosepromoter which is completely repressed in the absence of arabinose,allows cultures of transformed organisms to be expanded without biasingthe polyclonal antibody population for antibodies that are bettertolerated by E. coli until induction.

A variant of this vector was also constructed to clone any protein withor without a signal sequence. The modified vector has the NcoIrestriction site and all of the pelB signal-sequence removed. In itsplace a NsiI restriction site was incorporated such that upon NsiIdigestion and then T4 digestion, there is single base added, in frame,to the araBAD promoter that becomes the adenosine residue (A) of the ATGinitiation codon. The HindIII site and restoration of the tetracyclinepromoter with primer J (Table 3) remains the same as described for thepBRncoH3 vector. Additionally, the T4 exonuclease cloning process isidentical to that described above, except that the 5′ PCR primer used toamplify the insert contains 20 bp of vector sequence at its 5′-endcorresponding to 3′-end of the araBAD promoter rather than the 3′-end ofthe PelB signal sequence.

Three PCR primers, K, L, and M (Table 3) were used for amplifying thearaC regulatory gene (including the araBAD promoter). The 5′-primer,primer K, includes an EcoRI restriction site at its 5′-end for ligatingthe ara insert into the pBR vector. The 3′-end of the insert wasamplified using two primers because a single primer would have been toolarge to synthesize. The inner 3′-primer (L) introduces the NsiIrestriction site, in frame, with the araBAD promoter, with the outer 3′primer (M) introducing the HindIII restriction site that will be usedfor ligating the insert into the vector.

The PCR reaction was performed as in Example 3 on a 4×100 μl scale; thereactions containing 100 pmol of 5′ primer (K), 1 pmol of the inner 3′primer (L), and 100 pmol of outer 3′ primer (M), 10 μl 2 mM dNTPs, 0.5μL Taq DNA Polymerase, 10 μl 10×Taq DNA polymerase buffer with MgCl₂,and H₂O to 100 μL. The araCparaBAD PCR product was precipitated andfractionated by agarose gel electrophoresis and full-length productsexcised from the gel, purified, resuspended in water, and prepared forcloning by digestion with EcoRI and HindIII as described earlier. ThepBR vector (Life Technologies, Gaithersburg, Md.) was prepared toreceive this insert by digestion with EcoRI and HindIII and purificationby agarose gel electrophoresis as described above.

The insert and vector were mixed (2:1 molar ratio) together with 1 μl 10mM ATP, 1 μl (1 U/μl) T4 DNA ligase, 1 μl 10×ligase buffer in a finalvolume of 10 μl and ligated overnight at 15° C. The ligation reactionwas diluted to 20 μl, and 1 μl electroporated into electrocompetent E.coli strain, DH10B (Example 8), plated on LB tetracycline (10 μg/ml)plates and grown overnight at 37° C. Clones were picked and grownovernight in 3 ml LB broth supplemented with tetracycline.

These clones were tested for the correct insert by PCR amplification(Example 3) with primers K and M, using 1 μl of overnight culture astemplate. Agarose gel electrophoresis of the PCR reactions demonstratedthat all clones had the araCparaB insert. The vector (plasmid) waspurified from each culture by Wizard miniprep columns followingmanufacturers recommendations. The new vector, pBRnsi contained the araCgene, the araBAD promoter, and a NsiI restriction site.

The vector was tested for expression by introducing a murine Fab. Theregion was amplified by PCR (Example 3) from a plasmid (20 ng)containing a murine Fab with primers O and N (Table 3). The primers, inaddition to having sequence specific to the Fab, contain 20 bp of vectorsequence at their 5′-end corresponding to the 3′-end araBAD promoter andthe 5′-end of the C_(H)1 region for cloning purposes. The pBRnsi vectorwas linearized with NsiI and HindIII. The vector and the PCR productwere run on an agarose gel, and full-length products were excised fromthe gel and purified as described previously. The vector and insert weredigested with T4 DNA polymerase and annealed as described earlier. Theannealed DNA (1 μl) was electroporated (Example 8) into 30 μl ofelectrocompetent E. coli strain DH10B. The transformed cells werediluted to 1.0 ml with 2×YT broth and 1 μl, 10 μl, and 100 μl plated onLB agar plates supplemented with tetracycline (10 μg/ml) and grownovernight at 37° C.

Nitrocellulose lifts were placed on the placed on the surface of theagar plates for 1 min and processed as described (Section 12.24,Molecular Cloning, A laboratory Manual, (1989) Sambrook. J.). Thefilters were developed with goat anti-kappa-AP, and a positive (kappaexpressing) clone was picked and grown overnight in 2×YT (10 μg/mltetracycline) at 37° C. The vector (plasmid) was purified from theculture by Wizard miniprep columns (Promega, Madison, Wis.) followingmanufacturers recommendations. The Fab region was excised byNcoI/HindIII digestion and replaced with a stuffer fragment of unrelatedDNA by ligation as described above. The ligation reaction was diluted to20 μl, and 1 μl electroporated (Example 8) into electrocompetent E. colistrain DH10B, plated on LB ampicillin (100 μg/ml) and incubated at 37°C. After overnight incubation, transformants were picked and grownovernight in LB broth supplemented with ampicillin (100 μg/ml). Thevector (plasmid) was purified from each culture by Wizard miniprepcolumns following manufacturers recommendations. This modified vector,pBRnsiH3, is tet sensitive, but still retains ampicillin resistance forgrowing preparations of the vector.

EXAMPLE 15 Subcloning Polyclonal Fab Populations into Expression Vectorsand Electroporation into Escherichia coli

The polyclonal IL8 antibody phage form both the 10⁹ and 10¹⁰ affinitycuts (see Example 13) were diluted 1/30 in 2×YT and 1 μl used astemplate for PCR amplification of the antibody gene inserts with primers197 (Example 5) and 970 (see below). PCR (3-100 μL reactions) wasperformed using a high-fidelity PCR system, Expand (Roche MolecularBiochemicals, Indianapolis, Ind.) to minimize errors incorporated intothe DNA product. Each 100 μl reaction contained 100 pmol of 5′ primer197, 100 pmol of 3′ primer 970, 0.7 units of Expand DNA polymerase, 10μl 2 mM dNTPs, 10 μl 10×Expand reaction buffer, 1 μl diluted phage stockas template, and water to 100 μl. The reaction was carried out in aPerkin-Elmer thermal cycler (Model 9600) using the following thermalprofile: one cycle of denaturation at 94° C. (1 min); ten cycles ofdenaturation (15 sec, 94° C.), annealing (30 sec, 55° C.), elongation(60 sec, 72° C.); fifteen cycles of denaturation (15 sec, 94° C.),annealing (30 sec, 55° C.), elongation (80 sec plus 20 sec for eachadditional cycle, 72° C.); elongation (6 min, 72° C.); soak (4° C.,indefinitely). The PCR products were ethanol precipitated, pelleted anddried as described above. The DNA was dissolved in water andfractionated by agarose gel electrophoresis. Only full-length productswere excised from the gel, purified, and resuspended in water asdescribed earlier.

Primer 970—5′ GT GAT AAA CTA CCG TA AAG CTT ATC GAT GAT AAG CTG TCA ATTA GTG ATG GTG ATG GTG ATG AGA TTT G (SEQ ID NO:29)

The insert and NcoI/HindIII digested pBRncoH3 vector were prepared forT4 exonuclease digestion by adding 1.0 μl of 10×Buffer A to 1.0 μg ofDNA and bringing the final volume to 9 μl with water. The samples weredigested for 4 min at 30° C. with 1 μl (1U/μl) of T4 DNA polymerase. TheT4 DNA polymerase was heat inactivated by incubation at 70° C. for 10min. The samples were cooled, briefly spun, and 100 ng of the digestedantibody gene insert and 1 μl of 10×annealing buffer were mixed with 100ng of digested vector in a 1.5 mL tube. The volume was brought to 10 μlwith water, heated to 70° C. for 2 min and cooled over 20 min to roomtemperature to allow the insert and vector to anneal. The insert andvector were ligated together by adding 1 μl of 10×synthesis buffer, 1 μlT4 DNA ligase (1U/μl), 1 μl diluted T7 DNA polymerase (1U/μl) andincubating at 37° C. for 15 min.

The ligated DNA (1 μl) was diluted into 2 μL of water, then 1 μL of thediluted DNA was electroporated (Example 8) into 40 μl ofelectrocompetent E. coli strain, DH10B. The transformed cells werediluted to 1.0 ml with 2×YT broth and 10 μl of 10⁻¹, 10⁻² and 10⁻³dilutions plated on LB agar plates supplemented with tetracycline at 10μg/ml to determine the size of the subcloned polyclonal population. The10⁹ affinity polyclonal had approximately 6000 different clones, and the10¹⁰ affinity polyclonal had approximately 10,000 different clones. Theremaining cells were incubated at 37° C., 300 rpm for 1 hr, and then theentire culture was transferred into 50 ml 2×YT supplemented with 1%glycerol and 20 μg/ml tetracycline and grown overnight at 37° C. Theovernight culture was diluted 1/100 into the same media, grown 8 hr, andglycerol freezer stocks made for long term storage at −80° C.

Monoclonal antibodies were obtained by picking individual colonies offthe LB agar plates supplemented with tetracycline used to measure thesubcloning efficiency or from plates streaked with cells from theglycerol freezer stocks. The picks were incubated overnight at 37° C.,300 rpm in a shake flask containing 2×YT media and 10 μg/mL tetracyclin.Glycerol freezer stocks were made for each monoclonal for long termstorage at −80° C. A total of 15 different colonies were picked off ofthe 10⁹ affinity cut and analyzed for binding to IL8. Of those 15clones, two expressed a very low amount of antibody, one expressedantibody but did not bind IL8, two expressed functional antibody but theDNA sequence was ambiguous most likely due to sequence template quality,and one expressed functional protein but was not sequenced. Nine cloneswere sequenced as described in Example 22. A total of 21 differentcolonies were picked off of the 10¹⁰ affinity cut and analyzed forbinding to IL8. Of those 21 clones, four expressed a very low amount ofantibody, three expressed antibody but did not bind IL8, and fourexpressed functional protein but were not sequenced. Ten clones weresequenced as described in Example 22.

EXAMPLE 16 Expression of IL8 or Antibodies in Shake Flasks andPurification

A shake flask inoculum is generated overnight from a −80° C. cell bankor from a colony (Example 15) in an incubator shaker set at 37° C., 300rpm. The cells are cultured in a defined medium described above. Theinoculum is used to seed a 2 μL Tunair shake flask (Shelton Scientific,Shelton, Conn.) which is grown at 37° C., 300 rpm. Expression is inducedby addition of L(+)-arabinose to 2 g/L during the logarithmic growthphase, following which, the flask is maintained at 23° C., 300 rpm.Following batch termination, the culture is passed through an M-110YMicrofluidizer (Microfluidics, Newton, Mass.) at 17000 psi.

Purification employs immobilized metal affinity chromatography.Chelating Sepharose FastFlow resin (Pharmacia, Piscataway, N.J.) ischarged with 0.1 M NiCl₂ and equilibrated in 20 mM borate, 150 mM NaCl,10 mM imidazole, 0.01% NaN₃, pH 8.0 buffer. A stock solution is used tobring the culture to 10 mM imidazole. The supernatant is then mixed withthe resin and incubated for at least 1 hour in the incubator shaker setat room temperature, 150-200 rpm. IL8 or antibody is captured by meansof the high affinity interaction between nickel and the hexahistidinetag on the protein. After the batch binding is complete, the resin isallowed to settle to the bottom of the bottle for at least 10 min. Theculture is carefully poured out of the bottle, making sure that theresin is not lost. The remaining culture and resin mixture is pouredinto a chromatography column. After washing, the protein is eluted with20 mM borate, 150 mM NaCl, 200 mM imidazole, 0.01% NaN₃, pH 8.0 buffer.If needed, the protein pool is concentrated in a Centriprep-10concentrator (Amicon, Beverly, Mass.) at 3500 rpm. It is then dialyzedovernight into 20 mM borate, 150 mM NaCl, 0.01% NaN₃, pH 8.0 forstorage, using 12-14,000 MWCO dialysis tubing.

IL8 was furthur purified by the following procedure. The protein wasdialyzed exhaustively against 10 mM sodium phosphate, 150 mM sodiumchloride, pH 7.35, and diluted 1:3 with 10 mM sodium phosphate, pH 7.35.This material was loaded onto a Q-Sepharose column (Amersham PharmaciaBiotech, Piscataway, N.J.) equilibrated in 10 mM sodium phosphate, 40 mMNaCl. The IL8 was contained in the flow through fraction. BySDS-polyacrylamide gel analysis, the IL8 was greater than 95% pure. TheIL8 was brought to 120 mM NaCl and 0.01% NaN₃ and stored at −80° C.

EXAMPLE 17 Preparation of 7F11 Monoclonal Antibody

Synthesis of Acetylthiopropionic Acid

To a stirred solution of 3-mercaptopropionic acid (7 ml, 0.08 moles) andimidazole (5.4 g, 0.08 moles) in tetrahydrofuran (THF, 700 ml) was addeddropwise over 15 min, under argon, a solution of 1-acetylimidazole (9.6g, 0.087 moles) in THF (100 ml). The solution was allowed to stir afurther 3 hr at room temperature after which time the THF was removed invacuo. The residue was treated with ice-cold water (18 ml) and theresulting solution acidified with ice-cold concentrated HCl (14.5 ml) topH 1.5-2. The mixture was extracted with water (2×50 ml), dried overmagnesium sulfate and evaporated. The residual crude yellow oily solidproduct (10.5 g) was recrystallized from chloroform-hexane to afford 4.8g (41% yield) acetylthiopropionic acid as a white solid with a meltingpoint of 44-45° C.

Decapeptide Derivatives

The decapeptide, YPYDVPDYAS, (SEQ ID NO:30), (Chiron Mimotopes PeptideSystems, San Diego, Calif.) was dissolved (0.3 g) in dry DMF (5.4 mL) ina round bottom flask under argon with moderate stirring. Imidazole (0.02g) was added to the stirring solution. Separately, acetylthiopropionicacid (0.041 g) was dissolved in 0.55 mL of dry DMF in a round bottomflask with stirring and 0.056 g of 1,1′-carbonyldiimidazole (AldrichChemical Co., Milwaukee, Wis.) was added to the stirring solution. Theflask was sealed under argon and stirred for at least 30 min at roomtemperature. This solution was added to the decapeptide solution and thereaction mixture was stirred for at least six hr at room temperaturebefore the solvent was removed in vacuo. The residue in the flask wastriturated twice using 10 mL of diethyl ether each time and the etherwas decanted. Methylene chloride (20 mL) was added to the residue in theflask and the solid was scraped from the flask and filtered using a finefritted Buchner funnel. The solid was washed with an additional 20 mL ofmethylene chloride and the Buchner funnel was dried under vacuum. Inorder to hydrolyze the derivative to generate a free thiol, it wasdissolved in 70% DMF and 1 M potassium hydroxide was added to a finalconcentration of 0.2 M while mixing vigorously. The derivative solutionwas allowed to stand for 5 min at room temperature prior toneutralization of the solution by the addition of a solution containing0.5 M potassium phosphate, 0.1 M borate, pH 7.0, to which concentratedhydrochloric acid has been added to a final concentration of 1 M. Thethiol concentration of the hydrolyzed decapeptide derivative wasdetermined by diluting 10 μL of the solution into 990 μL of a solutioncontaining 0.25 mM 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB, AldrichChemical Co., Milwaukee Wis.) and 0.2 M potassium borate, pH 8.0. Thethiol concentration in mM units was equal to the A412(100/13.76).

Preparation of Conjugates of Decapeptide Derivative with Keyhole LimpetHemocyanin and Bovine Serum Albumin

Keyhole limpet hemocyanin (KLH, 6 ml of 14 mg/ml, Calbiochem, San Diego,Calif.) was reacted with sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SULFO-SMCC) by adding 15mg of SULFO-SMCC and maintaining the pH between 7 and 7.5 with 1Npotassium hydroxide over a period of one hr at room temperature whilestirring. The protein was separated from the unreacted SULFO-SMCC by gelfiltration chromatography in 0.1 M potassium phosphate, 0.02 M potassiumborate, and 0.15 M sodium chloride, pH 7.0, and 24 ml of KLH-maleimidewas collected at a concentration of 3.1 mg/ml. The hydrolyzeddecapeptide derivative was separately added to portions of theKLH-maleimide in substantial molar excess over the estimated maleimideamounts present and the solution was stirred for 4 hr at 4° C. and theneach was dialyzed against 3 volumes of one liter of pyrogen-freephosphate-buffered saline, pH7.4, prior to immunization.

Bovine serum albumin (BSA, 3.5 ml of 20 mg/ml) was reacted with SMCC byadding a solution of 6.7 mg of SMCC in 0.3 ml acetonitrile and stirringthe solution for one hr at room temperature while maintaining the pHbetween 7 and 7.5 with 1N potassium hydroxide. The protein was separatedfrom unreacted materials by gel filtration chromatography in 0.1 Mpotassium phosphate, 0.02 M potassium borate, 0.15 M sodium chloride, pH7.0. The hydrolyzed decapeptide derivative was separately added toportions of the BSA-maleimide in substantial molar excess over theestimated maleimide amounts present and the solution was stirred for 4hr at 4° C. The solutions were used to coat microtiter plates for thedetection of antibodies that bound to the decapeptide derivative bystandard techniques.

Production and Primary Selection of Monoclonal Antibodies

Immunization of Balb/c mice was performed according to the method ofLiu, et al. Clin Chem 25:527-538 (1987). Fusions of spleen cells withSP2/0-Ag 14 myeloma cells, propagation of hybridomas, and cloning wereperformed by standard techniques. Selection of hybridomas for furthercloning began with culture supernatant at the 96-well stage. A standardELISA procedure was performed with a BSA conjugate of decapeptidederivative adsorbed to the ELISA plate. Typically, a single fusion wasplated out in twenty plates and approximately 10-20 wells per plate werepositive by the ELISA assay. At this stage, a secondary selection couldbe performed if antibodies to the SMCC part of the linking arm were tobe eliminated from further consideration. An ELISA assay using BSAderivatized with SMCC but not linked to the decapeptide derivativeidentified which of the positive clones that bound the BSA conjugateswere actually binding the SMCC-BSA. The antibodies specific for SMCC-BSAmay be eliminated at this step. Monoclonal antibody 7F11, specific forthe decapeptide derivative, was produced and selected by this process.

EXAMPLE 18 Preparation of 7F11 Magnetic Latex

MAG/CM-BSA

To 6 mL of 5% magnetic latex (MAG/CM, 740 μm 5.0%, Seradyn,Indianapolis, Ind.) was added 21 mL of water followed by 3 mL of 600 mM2-(4-morpholino)-ethane sulfonic acid, pH 5.9 (MES, Fisher Scientific,Pittsburgh, Pa.). Homocysteine thiolactone hydrochloride (HCTL, 480 mg,Aldrich Chemical Co., Milwaukee, Wis.) and1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide (EDAC, 660 mg, AldrichChemical Co., Milwaukee, Wis.) were added in succession, and thereaction mixture was rocked at room temperature for 2 h. The derivatizedmagnetic latex was washed 3 times with 30 mL of water (with magnet as inExample 14) using probe sonication to resuspend the particles. Thewashed particles were resuspended in 30 mL of water. Three mL of asolution containing sodium hydroxide (2M) and EDTA (1 mM) was added tothe magnetic latex-HCTL suspension, and the reaction proceeded at roomtemperature for 5 min. The pH was adjusted to 6.9 with 6.45 mL of 1 Mhydrochloric acid in 500 mM sodium phosphate, 100 mM sodium borate. Thehydrolyzed magnetic latex-HCTL was separated from the supernate with theaid of a magnet, and then resuspended in 33 mL of 50 mM sodiumphosphate, 10 mM sodium borate, 0.1 mM EDTA, pH 7.0. The magnetic latexsuspension was then added to 2 mL of 36 mg mL-1 BSA-SMCC (made asdescribed in Example 21 with a 5-fold molar excess of SMCC over BSA),and the reaction mixture was rocked overnight at room temperature.N-Hydroxyethylmaleimide (NHEM, 0.42 mL of 500 mM, Organix Inc., Woburn,Mass.) was added to cap any remaining thiols for 30 min. After 30 min,the magnetic latex-BSA was washed twice with 30 mL of 50 mM potassiumphosphate, 10 mM potassium borate, 150 mM sodium chloride, pH 7.0(50/10/150) and twice with 30 mL of 10 mM potassium phosphate, 2 mMpotassium borate, 200 mM sodium thiocyanate, pH 7.0 (10/2/200). Themagnetic latex-BSA was resuspended in 30 mL of 10/2/200.

7F11-SH (1:5)

To a solution of 7F11 (3.8 mL of 5.85 mg mL⁻¹) was added 18 μL of SPDP(40 mM in acetonitrile). The reaction proceeded at room temperature for90 min after which taurine (Aldrich Chemical Co., Milwaukee, Wis.) wasadded to a final concentration of 20 mM. Fifteen min later DTT was addedto a final concentration of 2 mM, and the reduction reaction proceededat room temperature for 30 min. The 7F11-SH was purified on G-50 (40 mL)that was eluted with 50/10/150 plus 0.1 mM EDTA. The pool of purified7F11-SH was reserved for coupling to the MAG/CM-BSA-SMCC.

MAG/CM-BSA-7F11

SMCC (10 mg) was dissolved in 0.5 mL of dry dimethylformamide (AldrichChemical Co., Milwaukee, Wis.), and this solution was added to themagnetic latex-BSA suspension. The reaction proceeded at roomtemperature with gentle rocking for 2 h. Taurine was added to a finalconcentration of 20 mM. After 20 min the magnetic latex-BSA-SMCC wasseparated from the supernate with the aid of a magnet and thenresuspended in 10/2/200 (20 mL) with probe sonication. The magneticlatex was purified on a column of Superflow-6 (240 mL, SterogeneBioseparations Inc., Carlsbad, Calif.) that was eluted with 10/2/200.The buffer was removed, and to the magnetic latex cake was added 30 mLof 0.7 mg mL⁻¹ 7F11-SH. The reaction mixture was rocked overnight atroom temperature. After 20 hr the reaction was quenched withmercaptoethanol (2 mM, Aldrich Chemical Co., Milwaukee, Wis.) followedby NHEM (6 mM). The MAG/CM-7F11 was washed with 10/2/200 followed by50/10/150. The magnetic latex was then resuspended in 30 mL of50/10/150.

EXAMPLE 19 Cloning of the Mature Human Interleukin-8 Antigen

PCR primers A and B (5′ and 3′ respectively, Table 3) were madecorresponding to the coding sequence at the 5′-end of the mature humaninterleukin-8 antigen and the coding sequence at the 3′-end of humaninterleukin-8 (Genbank accession number M28130). The 5′ primer contains20 base pairs of vector sequence at its 5′-end corresponding to the3′-end of the pBRncoH3 vector (Example 14). The 3′ primer has sixhistidine codons inserted between the end of the coding sequence and thestop codon to assist in purification of the recombinant protein bymetal-chelate chromatography. The 3′ primer also has 19 base-pairs oftet promoter removed from the tet resistance gene in pBRncoH3 by HindIIIdigestion, and base-pairs of vector sequence 3′ to the HindIII site atits 5′ end (Example 14).

The PCR amplification of the mature interleukin-8 gene insert was doneon a 3×100 μl reaction scale each containing 100 pmol of 5′ primer (A),100 pmol of 3′ primer (B), 2.5 units of Expand polymerase, 10 μl 2 mMdNTPs, 10 μl 10×Expand reaction buffer, 1 μl of Clontech Quick-clonehuman liver cDNA (Clontech Laboratories, Palo Alto, Calif.) as template,and water to 100 μl. The reaction was carried out in a Perkin-Elmerthermal cycler as described in Example 18. The PCR products wereprecipitated and fractionated by agarose gel electrophoresis andfull-length products excised from the gel, purified, and resuspended inwater (Example 17). The insert and NcoI/HindIII digested pBRncoH3 vectorwere prepared for T4 exonuclease digestion by adding 1.0 μl of 10×BufferA to 1.0 μg of DNA and bringing the final volume to 9 μl with water. Thesamples were digested for 4 minutes at 30° C. with 1 μl (1U/μl) of T4DNA polymerase. The T4 DNA polymerase was heat inactivated by incubationat 70° C. for 10 minutes. The samples were cooled, briefly spun, and 15ng of the digested insert added to 100 ng of digested pBRncoH3 vector ina fresh microfuge tube. After the addition of 1.0 μl of 10×annealingbuffer, the volume was brought to 10μl with water. The mixture washeated to 70° C. for 2 minutes and cooled over 20 minutes to roomtemperature, allowing the insert and vector to anneal. The annealed DNAwas diluted one to four with distilled water and electroporated (example8) into 30μl of electrocompetent E. coli strain, DH10B. The transformedcells were diluted to 1.0 ml with 2×YT broth and 10 μl, 100 μl, 300 μlplated on LB agar plates supplemented with tetracycline (10 μg/ml) andgrown overnight at 37° C. Colonies were picked and grown overnight in2×YT (20 μg/ml tetracycline at 37° C. The following day glycerol freezerstocks were made for long term storage at −80° C. The sequence of theseclones was verified at MacConnell Research (San Diego, Calif.) by thedideoxy chain termination method using a Sequatherm sequencing kit(Epicenter Technologies, Madison, Wis.), oligonucleotide primers C and D(Table 3) that bind on the 5′ and 3′ side of the insert in the pBRvector, respectively, and a LI-COR 4000L automated sequencer (LI-COR,Lincoln, Nev.).

Table 3. PCR and Sequencing Primer Sequences (SEQ ID NOS:31, 32, 33 and34 respectively)

TABLE 3 PCR and Sequencing Primer Sequences (SEQ ID NOS: 31, 32, 33 and34 respectively) A- 5′ (TCGCTGCCCAACCAGCCATGGCCAGTGCTAAAGAACTTAGATCTCAG)B- 5′ (GTGATAAACTACCGCATTAAAGCTTATCGATGATAAGCTGTCAATTAGTGAT      GGTGATGGTGATGTGAATTCTCAGCCCTCTTCAA) C- 5′ (GCAACTCTCTACTGTTTCTCC)D- 5′ (GAGGATGACGATGAGCGC)

EXAMPLE 20 Estimation of Library Diversity

Upon the completion of library selection for a given target antigen, thelibrary contains members encoding antibodies exhibiting an affinitydetermined by the criteria used during the selection process.Preferably, the selection process is repeated until the majority of themembers in the library encode antibodies exhibiting the desiredcharacteristics. Most preferably, the selection process is repeateduntil substantially all of the members of the library encode antibodiesthat exhibit the desired affinity for the target antigen. In order toestimate the number of different antibodies in the selected library,individual members are randomly chosen and sequenced to determine iftheir amino acid sequences are different. Antibodies exhibiting at leastone amino acid difference in either the heavy or light chain variabledomain (preferably in the CDRs) are considered different antibodies. Arandom sampling of the library in such a manner provides an estimate ofthe frequency antibody copies in the library. If ten antibodies arerandomly sampled and each antibody amino acid sequence is distinct fromthe other sampled antibodies, then an estimate of 1/10 can be applied tothe frequency that one might expect to observe for repeated antibodiesin the library. A library with hundreds or thousands of total memberswill exhibit a probability distribution for the frequency of antibodycopies that closely approximates the Poisson distribution,Pr(y)=e^(−η)η^(y)/y!, where the probability of a particular value y ofthe frequency is dependent only on the mean frequency η. If no antibodyreplicates are observed in a random sampling of ten antibodies, then anestimate for η is 0.1 and the probability of not observing a copy of alibrary member when randomly sampling the library is estimated byPr(0)=e^(−0.1)=0.9. Multiplying this probability by the total number ofmembers in the library provides an estimate of the total number ofdifferent antibodies in the library.

EXAMPLE 21 Determination of Antibody Affinity for IL-8 Labeled withBiotin

The equilibrium binding constants of individual monoclonal antibodieswere determined by analysis of the total and free antibodyconcentrations after a binding equilibrium was established in thepresence of biotinylated IL-8 at 10⁻¹⁰ M in a 1% solution of bovineserum albumin buffered at pH 8.0. In all experiments the antibody wasmixed with IL-8 and incubated overnight at room temperature before thebiotin-labeled IL-8 was removed from the solution by addingsuperparamagnetic microparticles (0.96 μm, Bangs Laboratories, Carmel,Ind.) coated with NeutrAvidin™ (deglycosylated avidin, Pierce, Rockford,Ill.) incubating for 10 minutes, and separating the particles from thesolution using a permanent magnet. The supernatant solution was removedfrom the microtiter wells containing the magnetic particles and theantibody concentration was determined. The concentration of totalantibody added to the individual wells was determined by quantifying theantibody in a sample that was not mixed with IL-8. The concentration ofimmunoreactive antibody (the fraction of the antibody protein that wascapable of binding to IL-8) was determined by incubating a large excessof biotin-labeled IL-8 with a known concentration of antibody for asufficient time to reach equilibrium, removing the IL-8 using magneticlatex as described above, and quantifying the concentration of antibodyleft in the solution using the assay described below. The fraction ofantibody that bound to the excess of IL-8 is the immunoreactive fractionand the fraction that did not bind to IL-8 is the non-immunoreactivefraction. When determining the concentration of total antibody in anequilibrium mixture, the antibody concentration is the amount of totalantibody in the mixture determined from the assay described belowmultiplied by the immunoreactive fraction. Similarly, when calculatingthe free antibody in an equilibrium mixture after the removal of IL-8,the non-immunoreactive fraction of antibody is subtracted from the freeantibody concentration determined by the assay described below. Thebound fraction, B, is determined by subtracting the free immunoreactiveantibody concentration in the mixture, F, from the total immunoreactiveantibody concentration in the mixture. From the Law of Mass Action,B/F=−KB+KT where T is the total antigen concentration. A plot of B/F vs.B yields a slope of −K and a y-intercept of KT.

To determine the antibody concentrations in samples a sandwich assay wasconstructed using immobilized monoclonal antibody 7F11 to bind thedecapeptide tag present a the C-terminus of the kappa chain andaffinity-purified goat-anti-human kappa antibody conjugated to alkalinephosphatase (Southern Biotechnology Associates, Birmingham, Ala.) tobind the kappa chain of each human antibody. A purified antibody ofknown concentration with the same kappa chain construction as theassayed antibodies was used to calibrate the assay. The 7F11 antibodywas labeled with biotin and immobilized on microtiter plates coated withstreptavidin using standard methods. The assay was performed by adding50 μl of sample from the equilibrium mixtures to each well andincubating for four hours at room temperature. The conjugate was addedat a final concentration of approximately 0.125 μg/ml to each well andincubated overnight at room temperature. The wells were washed using anautomatic plate washer with borate buffered saline containing 0.02%polyoxyethylene 20-sorbitan monolaurate at pH 8.2 and the ELISAAmplification System (Life Technologies, Gaithersburg, Md.) was employedto develop the assay. The absorbance at 490 nm was measured using amicrotiter plate reader and the unknown antibody concentrations weredetermined from the standard curve.

TABLE 4 Monoclonal Antibody % Immunoreactive Protein Affinity (10¹⁰ M⁻¹)M1-3 93 6.1 M1-4 93 22 M1-5 90 11 M1-8 91 10 M1-10 90 6.1 M1-21 67 6.6M1-23 91 8.9 M1-25 90 6.4 M2-11 93 10 M2-12 93 28 M2-16 90 1.9 M2-18 805.4 M2-20 94 37 M2-34 94 27

EXAMPLE 22 DNA Sequence Analysis of Random Clones

The glycerol freezer stocks (Example 15) corresponding to eachmonoclonal Fab to be analyzed were used to inoculate 50 ml cultures forplasmid isolation and subsequent DNA sequencing of the interleukin-8insert. After overnight growth in 2×YT (10 μg/ml tetracycline) at 37°C., the recombinant plasmid was purified using a Qiagen Plasmid Midi kit(Qiagen, Valencia, Calif.) following manufacturer's recommendations. Thesequence corresponding to the kappa and heavy chain variable andconstant regions for each monoclonal was determined at MacConnellResearch (San Diego, Calif.). The nomenclature used for antibodies isthe same as that in Example 21. Sequencing was done by the dideoxy chaintermination method using a Sequatherm sequencing kit (EpicenterTechnologies, Madison, Wis.), oligonucleotide primers C and D (Table 3)that bind on the 5′ and 3′ side of the Fab cassette in the pBR vector,respectively, and a LI-COR 4000L automated sequencer (LI-COR, Lincoln,Nev.).

M1-1L (SEQ ID NO:35)

AAATTGTGTTGACGCATTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGGGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGAACTGGCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTCTTATCCATATGATGTGCCAGATTATGCGAGC

M1-3L (SEQ ID NO:37)

GAAATAGTGATGACGCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCTCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTCTTATCCATATGATGTGCCAGATTATGCGAGC

M1-4L (SEQ ID NO:39)

GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCACATCTATGGTGCATCCAGAAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTTTGGTAGCTCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTCTTATCCATATGATGTGCCAGATTATGCGAGC

M1-5L (SEQ ID NO:41)

GAAATAGTGATGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCTATATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTCTTATCCATATGATGTGCCAGATTATGCGAGC

M1-8L (SEQ ID NO:43)

GAAATAGTGATGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCACCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGTTAGCTCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTCTTATCCATATGATGTGCCAGATTATGCGAGC

M1-10L (SEQ ID NO:45)

GATGTTGTGATGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCCCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTCTTATCCATATGATGTGCCAGATTATGCGAGC

M1-21L (SEQ ID NO:47)

GCCATCCGGATGACCCAGTCTCCATCCTTCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGTCAGTGGATCTGGGACAGATCTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAGTGTGGTTACAGTACACCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTCTTATCCATATGATGTGCCAGATTATGCGAGC

M1-23L (SEQ ID NO:49)

GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCTCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAGGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTCTTATCCATATGATGTGCCAGATTATGCGAGC

M1-25L (SEQ ID NO:51)

GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAAACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTCTTATCCATATGATGTGCCAGATTATGCGAGC

M1-1H (SEQ ID NO:53)

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAAGTCCCTGAGACTCTCCTGTGCAGCGTCTGAATTCACCATCAGTTACTATGGCATGCACTGGGTCCGCCAGGTTCCAGGCAAGGGGCTGGAGTGGGTGGCAGCTGTCTGGTATGATGAAAGTACTACATATTCTCCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACGATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATAGGGTGGGCCTCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGCAGAGCCCAAATCTCATCACCATCACCATCAC

M1-3H (SEQ ID NO:55)

CCGATGTGCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTTACTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGACACTTATAACCTATGATGGAGATAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGACGGGATCGGGTACTTTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGCAGAGCCCAAATCTCATCACCATCACCATCAC

M1-4H (SEQ ID NO:57)

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAAGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTTACTATGGCATGCACTGGGTCCGCCAGGTTCCAGGCAAGGGGCTGGAGTGGGTGGCAGCTGTCTGGTATGATGGAAGTACTACATATTCTCCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACGATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATAGGGTGGGCCTCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGCAGGGCCCAAATCTCATCACCATCACCATCAC

M1-5H (SEQ ID NO:59)

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTTACCTTCAGTTACTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGACACTTATAACCTATGATGGAGATAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGACGGGATCGGGTACTTTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGCAGAGCCCAAATCTCATCACCATCACCATCAC

M1-8H (SEQ ID NO:61)

CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAAGTCCCTGAAACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTTACTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGCTGTATGGTATGATGGAAGTAACACATACTCTCCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACGATTCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATAGGGTGGGCCTCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGCAGAGCCCAAATCTCATCACCATCACCATCAC

M1-10H (SEQ ID NO:63)

CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACATCCTGGGGGGTCCCTGAGACTCTCCTGTGAAGGCTCTGGATTCATCTTCAGGAACCATCCTATACACTGGGTTCGCCAGGCTCCAGGAAAAGGTCTGGAGTGGGTATCAGTTAGTGGTATTGGTGGTGACACATACTATGCAGACTCCGTGAAGGGCCGATTCTCCATCTCCAGAGACAATGCCAAGAACTCCTTGTATCTTCAAATGAACAGCCTGAGAGCCGAGGACATGGCTGTGTATTACTGTGCAAGAGAATATTACTATGGTTCGGGGAGTTATCGCGTTGACTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGCAGAGCCCAAATCTCATCACCATCACCATCAC

M1-2H (SEQ ID NO:65)

CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAAGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTTACTATGGCATGCACTGGGTCCGCCAGGTTCCAGGCAAGGGGCTGGAGTGGGTGGCAGCTGTCTGGTATGATGGAAGTACTACATATTCTCCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACGATTCCAAGAACACGCTGTATCTGCAAATGAGCAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATAGGGTGGGCCTCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGCAGAGCCCAAATCTCATCACCATCACCATCAC

M1-23H (SEQ ID NO:67)

CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTAACTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGCTATATGGTATGATGGAAGTAAAACATACAATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATGGGATAGGCTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGCAGAGCCCAAATCTCATCACCATCACCATCAC

M1-25H (SEQ ID NO:69)

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTTACTATGGCATGCACTGGGTCCGCCAGGTTCCAGGCAAGGGGCTGGAGTGGGTGGCAGCTGTCTGGTATGATGGAAGTACTACATATCCTCCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACGATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTTTATTACTGTGCGAGAGATAGGGTGGGCCTCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGCAGAGCCCAAATCTCATCACCATCACCATCAC

M2-11L (SEQ ID NO:71)

GAAATAGTGATGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGGGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCTCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAGATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTCTTATCCATATGATGTGCCAGATTATGCGAGC

M2-12L (SEQ ID NO:73)

GAAATAGTGATGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGGGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCTCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTCTTATCCATATGATGTGCCAGATTATGCGAGC

M2-16L (SEQ ID NO:75)

GAAATAGTGATGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGTCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTCTTATCCATATGATGTGCCAGATTATGCGAGC

M2-18L (SEQ ID NO:77)

GAAATAGTGATGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCACCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGTTAGCTCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTCTTATCCATATGATGTGCCAGATTATGCGAGC

M2-20L (SEQ ID NO:79)

GAAATAGTGATGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTACGGTGCATCCAGGAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCCATGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTCTTATCCATATGATGTGCCAGATTATGCGAGC

M2-31L (SEQ ID NO:81)

GAAATTGTGTTGACGCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTACGAACTGGCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTCTTATCCATATGATGTGCCAGATTATGCGAGC

M2-32L (SEQ ID NO:83)

GAAATTGTGTTGACGCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCGCTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAACGTAACAACTGGCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTCTTATCCATATGATGTGCCAGATTATGCGAGC

M2-33L (SEQ ID NO:85)

GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCTCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTTCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTCTTATCCATATGATGTGCCAGATTATGCGAGC

M2-34L (SEQ ID NO:87)

GAAATTGTGTTGACGCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTACGAACTGGCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTCTTATCCATATGATGTGCCAGATTATGCGAGC

M2-35L (SEQ ID NO:89)

GAAATTGTGTTGACGCAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTACGAACTGGCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTCTTATCCATATGATGTGCCAGATTATGCGAGC

M2-35L (SEQ ID NO:91)

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTTACCTTCAGTTACTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGACACTTATAACCTATGATGGAGATAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGACGGGATCGGGTACTTTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGCAGAGCCCAAATCTCATCACCATCACCATCAC

M2-12H (SEQ ID NO:93)

GATGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCATCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTTACCTTCAGTTACTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAATGGATGACACTTATATCCTATGATGGAGATAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGAAAATTCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGACGGGATCGGGTACTTTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAGCACCAAGGTGGACAAGAAAGCAGAGCCCAAATCTCATCACCATCACCATCAC

M2-16H (SEQ ID NO:95)

CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAAGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCAGCTTGAGTTACTATGGCATGCACTGGGTCCGCCAGGTTCCAGGCAAGGGGCTGGAGTGGGTGGCAGCTGTCTGGTATGATGGAAGTACTAGATATTCTCCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACGATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATAGGGTGGGCCTCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGCAGAGCCCAAATCTCATCACCATCACCATCAC

M2-18H (SEQ ID NO:97)

CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAAGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCAGCTTCAGTTACTATGGCATGCACTGGGTCCGCCAGGTTCCAGGCAAGGGGCTGGAGTGGGTGGCAGCTGTCTGGTATGATGGAAGTACTACATATTCTCCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACGATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATAGGGTGGGCCTCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGCAGAGCCCAAATCTCATCACCATCACCATCAC

M2-20H (SEQ ID NO:99)

CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGGCTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTTACTATGGTATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGTCACTTATAACATATGATGGAAGGAATAAATACTACGCCGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGAGAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAACTGAGGACACGGCTGAGTATTACTGTGCGAGAGACGGGATCGGATACTTTGACTACTGGGGCCAGGGAATCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAAGTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGCAGAGCCCAAATCTCATCACCATCACCATCAC

M2-31H (SEQ ID NO:101)

CAGGTGCAGCTGGTGGAGTCTGGGGGAGTCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACGTTCAGTTACTATGGTATACACTGGGTCCGCCAGGTTCCAGGCAAGGGACTAGAGTGGGTGGCACTTATATCATACGATGGAAGCAATAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACTCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGACTGGATCGGGTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGCAGAGCCCAAATCTCATCACCATCACCATCAC

M2-32H (SEQ ID NO:103)

CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACATCCTGGGGGGTCCCTGAGACTCTCCTGTGAAGGCTCTGGATTCATCTTCAGGAACCATCCTATACACTGGGTTCGCCAGGCTCCAGGAAAAGGTCTGGAGTGGGTATCAGTTAGTGGTATTGGTGGTGACACATACTATGCAGACTCCGTGAAGGGCCGATTCTCCATCTCCAGAGACAATGCCAAGAACTCCTTGTATCTTCAAATGAACAGCCTGAGAGCCGAGGACATGGCTGTGTATTACTGTGCAAGAGAATATTACTATGGTTCGGGGAGTTATCGCGTTGACTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGCAGAGCCCAAATCTCATCACCATCACCATCAC

M2-33H (SEQ ID NO:105)

CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTTACCTTCAGTTACTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAATGGATGACACTTATAACCTATGATGGAGATAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGACGGGATCGGGTACTTTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGCAGAGCCCAAATCTCATCACCATCACCATCAC

M2-34H (SEQ ID NO:107)

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACGTTCAGTTACTATGGTATACACTGGGTCCGCCAGGTTCCAGGCAAGGGACTAGAGTGGGTGGTACTTATATCATACGATGGAAGCAATAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACTCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGACTGGATCGGGTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGCAGAGCCCAAATCTCATCACCATCACCATCAC

M2-35H (SEQ ID NO:109)

CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACGATCAGTTACTATGGTATACACTGGGTCCGCCAGGTTCCAGGCAAGGGACTAGAGTGGGTGGAACTTATATCATACGATGGAAGCAATAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACTCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGACTGGATCGGGTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGCAGAGCCCAAATCTCATCACCATCACCATCAC

Translated amino acid sequences of sequenced antibodies. M1-H HeavyChain Variable and CH1 Regions 10-9M Affinity Cut (SEQ ID NOS: 64, 54,66, 68, 70, 56, 58, 60, and 62 repectively)1                                                   50 M1_10H QVQLVQSGGGLVHPGGSLRL SCEGSGFIFR NHPIHWVRQA PGKGLEWVSV  M1_1H QVQLVESGGG VVQPGKSLRLSCAASEFTIS YYGMHWVRQV PGKGLEWVAA M1_21H QVQLVQSGGG VVQPGKSLRL SCAASGFTFSYYGMHWVRQV PGKGLEWVAA M1_23H QVQLVQSGGG VVQPGRSLRL SCAASGFTFS NYGMHWVRQAPGKGLEWVAA M1_25H QVQLVESGGG LVQPGGSLRL SCAASGFTFS YYGMHWVRQV PGKGLEWVAA M1_3H DVQLVQSGGG VVQPGRSLRL SCAASGFTFS YYGMHWVRQA PGKGLEWVTL  M1_4HQVQLVESGGG VVQPGKSLRL SCAASGFTFS YYGMHWVRQV PGKGLEWVAA  M1_5H QVQLVESGGGVVQPGRSLRL SCAASGFTFS YYGMHWVRQA PGKGLEWVTL  M1_8H QVQLVQSGGG VVQPGKSLKLSCAASGFTFS YYGMHWVRQA PGKGLEWVAA51                                                 100 M1_10H SGIGGDTYY.ADSVKGRFSI SRDNAKNSLY LQMNSLRAED MAVYYCAREY  M1_1H VWYDESTTYS PDSVKGRFTISRDDSKNTLY LQMNSLRAED TAVYYCARDR M1_21H VWYDGSTTYS PDSVKGRFTI SRDDSKNTLYLQMSSLRAED TAVYYCARDR M1_23H IWYDGSKTYN ADSVKGRFTI SRDNSKNTLY LQMNSLRAEDTAVYYCARDG M1_25H VWYDGSTTYP PDSVKGRFTI SRDDSKNTLY LQMNSLRAED TAVYYCARDR M1_3H ITYDGDNKYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDG  M1_4HVWYDGSTTYS PDSVKGRFTI SRDDSKNTLY LQMNSLRAED TAVYYCARDR  M1_5H ITYDGDNKYYADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDG  M1_8H VWYDGSNTYS PDSVKGRFTISRDDSKNTVY LQMNSLRAED TAVYYCARDR101                                                150 M1_10H YYGSGSYRVDYYYYGMDVWG QGTTVTVSSA STKGPSVFPL APSSKSTSGG  M1_1H VG........ ....LFDYWGQGTLVTVSSA STKGPSVFPL APSSKSTSGG M1_21H VG........ ....LFDYWG QGTLVTVSSASTKGPSVFPL APSSKSTSGG M1_23H IG........ ....YFDYWG QGTLVTVSSA STKGPSVFPLAPSSKSTSGG M1_25H VG........ ....LFDYWG QGTLVTVSSA STKGPSVFPL APSSKSTSGG M1_3H IG........ ....YFDYWG QGTLVTVSSA STKGPSVFPL APSSKSTSGG  M1_4HVG........ ....LFDYWG QGTLVTVSSA STKGPSVFPL APSSKSTSGG  M1_5H IG............YFDYWG QGTLVTVSSA STKGPSVFPL APSSKSTSGG  M1_8H VG........ ....LFDYWGQGTLVTVSSA STKGPSVFPL APSSKSTSGG151                                                200 M1_10H TAALGCLVKDYFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV  M1_1H TAALGCLVKD YFPEPVTVSWNSGALTSGVH TFPAVLQSSG LYSLSSVVTV M1_21H TAALGCLVKD YFPEPVTVSW NSGALTSGVHTFPAVLQSSG LYSLSSVVTV M1_23H TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSGLYSLSSVVTV M1_25H TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV M1_3H TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV  M1_4HTAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV  M1_5H TAALGCLVKDYFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV  M1_8H TAALGCLVKD YFPEPVTVSWNSGALTSGVH TFPAVLQSSG LYSLSSVVTV201                                  237 M1_10H PSSSLGTQTY ICNVNHKPSNTKVDKKAEPK SHHHHHH  M1_1H PSSSLGTQTY ICNVNHKPSN TKVDKKAEPK SHHHHHHM1_21H PSSSLGTQTY ICNVNHKPSN TKVDKKAEPK SHHHHHH M1_23H PSSSLGTQTYICNVNHKPSN TKVDKKAEPK SHHHHHH M1_25H PSSSLGTQTY ICNVNHKPSN TKVDKKAEPKSHHHHHH  M1_3H PSSSLGTQTY ICNVNHKPSN TKVDKKAEPK SHHHHHH  M1_4HPSSSLGTQTY ICNVNHKPSN TKVDKKAGPK SHHHHHH  M1_5H PSSSLGTQTY ICNVNHKPSNTKVDKKAEPK SHHHHHH  M1_8H PSSSLGTQTY ICNVNHKPSN TKVDKKAEPK SHHHHHH M1-LKappa Chain Variable and Constant Regions 10-9 Affinity Cut (SEQ ID NOS:46, 36, 48, 50, 52, 38, 40, 42, and 44 respectively)1                                                   50 M1_10L DVVMTQSPATLSLSPGERAT LSCRASQSVS S.YLAWYQQK PGQAPRLLIY  M1_1L EIVLTQSPAT LSLSPGERATLSCRASQGVS S.YLAWYQQK PGQAPRLLIY M1_21L AIRMTQSPSF LSASVGDRVT ITCRASQSISS.YLNWYQQK PGKAPKLLIY M1_23L EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQKPGQAPRLLIY M1_25L EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY M1_3L EIVMTQSPAT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY  M1_4LEIVLTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLHIY  M1_5L EIVMTQSPGTLSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY  M1_8L EIVMTQSPGT LSLSPGERATLSCRASQSVS STYLAWYQQK PGQAPRLLIY51                                                 100 M1_10L DASNRATGIPARFSGSGSGT DFTLTISSLE PEDFAVYYCQ QRSNWP.PTF  M1_1L DASNRATGIP ARFSGSGSGTDFTLTISSLE PEDFAVYYCQ QRSNWP.RTF M1_21L AASSLQSGVP SRFSVSGSGT DLTLTISSLQPEDFATYYCQ CGYSTP.FTF M1_23L GASSRATGIP DRFSGSGSGT DFTLTISRLE PEDFAVYYCQQYGSSPPYTF M1_25L GASSRATGIP NRFSGSGSCT DFTLTISRLE PEDFAVYYCQ QYGSS..FTF M1_3L GASSRATGIP DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYGSSPPFTF  M1_4LGASRRATGIP DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QFGSS..FTF  M1_5L GASSRATGTPDRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYGSSPIFTF  M1_8L GASSRATGIP DRFSGSGSGTDFTLTTSRLE PEDFAVYYCQ QYVSS..FTF101                                                150 M1_10L GGGTKVEIKRTVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW  M1_1L GQGTKVEIKR TVAAPSVFIFPPSDEQLKSG TASVVCLLNN FYPREAKVQW M1_21L GPGTKVDIKR TVAAPSVFIF PPSDEQLKSGTASVVCLLNN FYPREAKVQW M1_23L GQGTKLEIKR TVAAPSVFIF PPSDEQLKSG TASVVCLLNNFYPREAKVQW M1_25L GPGTKVDIKR TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW M1_3L GPGTKVDIKR TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW  M1_4LGPGTKVDIKR TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW  M1_5L GPGTKVDIKRTVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW  M1_8L GPGTKVDIKR TVAAPSVFIFPPSDEQLKSG TASVVCLLNN FYPREAKVQW151                                                200 M1_10L KVDNALQSGNSQESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH  M1_1L KVDNALQSGN SQESVTEQDSKDSTYSLSST LTLSKADYEK HKVYACEVTH M1_21L KVDNALQSGN SQESVTEQDS KDSTYSLSSTLTLSKADYEK RKVYACEVTH M1_23L RVDNALQSGN SQESVTEQDS KDSTYSLSST LTLSKADYEKHKVYACEVTH M1_25L KVDNALQSGN SQESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH M1_3L KVDNALQSGN SQESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH  M1_4LKVDNALQSGN SQESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH  M1_5L KVDNALQSGNSQESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH  M1_8L KVDNALQSGN SQESVTEQDSKDSTYSLSST LTLSKADYEK HKVYACEVTH 201                      226 M1_10LQGLSSPVTKS FNRGESYPYD VPDYAS M1_1L QGLSSPVTKS FNRGESYPYD VPDYAS M1_21LQGLSSPVTKS FNRGESYPYD VPDYAS M1_23L QGLSSPVTKS FNRGESYPYD VPDYAS M1_25LQGLSSPVTKS FNRGESYPYD VPDYAN M1_3L QGLSSPVTKS FNRGESYPYD VPDYAS M1_4LQGLSSPVTKS FNRGESYPYD VPDYAS M1_5L QGLSSPVTKS FNRGESYPYD VPDYAS M1_8LQGLSSPVTKS FNRGESYPYD VPDYAS M2-H Heavy Chain VH-CH1 Sequence 10-10MAffinity Cut (SEQ ID NOS: 92, 94, 96, 98, 100, 102, 104, 106, 108, and110 respectively) 1                                                   50M2_11H QVQLVESGGG VVQPGRSLRL SCAASGFTFS YYGMHWVRQA PGKGLEWVTL M2_12HDVQLVESGGG VVHPGRSLRL SCAASGFTFS YYGMHWVRQA PGKGLEWMTL M2_16H QVQLVQSGGGVVQPGKSLRL SCAASGFSLS YYGMHWVRQV PGKGLEWVAA M2_18H QVQLVQSGGG VVQPGKSLRLSCAASGFSFS YYGMHWVRQV PGKGLEWVAA M2_20H QVQLVQSGGG VVQPGRSLRL SCAASGFTFSYYGMHWVRQA PGKGLEWVSL M2_31H QVQLVESGGV VVQPGRSLRL SCAASGFTFS YYGIHWVRQVPGKGLEWVAL M2_32H QVQLVQSGGG LVHPGGSLRL SCEGSGFIFR NHPIHWVRQA PGKGLEWVSVM2_33H QVQLVQSGGG VVQPGRSLRL SCAASGFTFS YYGMHWVRQA PGKGLEWMTL M2_34HQVQLVESGGG VVQPGRSLRL SCAASGFTFS YYGIHWVRQV PGKGLEWVVL M2_35H QVQLVESGGGVVQPGRSLRL SCAASGFTIS YYGIHWVRQV PGKGLEWVEL51                                                 100 M2_11H ITYDGDNKYYADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDG M2_12H ISYDGDNKYY ADSVKGRFTISRENSKNTLY LQMNSLRAED TAVYYCARDG M2_16H VWYDGSTRYS PDSVKGRFTI SRDDSKNTLYLQMNSLRAED TAVYYCARDR M2_18H VWYDGSTTYS PDSVKGRFTI SRDDSKNTLY LQMNSLRAEDTAVYYCARDR M2_20H ITYDGRNKYY ADSVKGRFTI SRENSKNTLY LQMNSLRTED TAEYYCARDGM2_31H ISYDGSNKYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDW M2_32HSGIGG.DTYY ADSVKGRFSI SRDNAKNSLY LQMNSLRAED MAVYYCAREY M2_33H ITYDGDNKYYADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARDG M2_34H ISYDGSNKYY ADSVKGRFTISRDNSKNTLY LQMNSLRAED TAVYYCARDW M2_35H ISYDGSNKYY ADSVKGRFTI SRDNSKNTLYLQMNSLRAED TAVYYCARDW101                                                150 M2_11H IG............YFDYWG QGTLVTVSSA STKGPSVFPL APSSKSTSGG M2_12H IG........ ....YFDYWGQGTLVTVSSA STKGPSVFPL APSSKSTSGG M2_16H VG........ ....LFDYWG QGTLVTVSSASTKGPSVFPL APSSKSTSGG M2_18H VG........ ....LFDYWG QGTLVTVSSA STKGPSVFPLAPSSKSTSGG M2_20H IG........ ....YFDYWG QGILVTVSSA STKGPSVFPL APSSKSTSGGM2_31H IG........ ....YFDYWG QGTLVTVSSA STKGPSVFPL APSSKSTSGG M2_32HYYGSGSYRVD YYYYGMDVWG QGTTVTVSSA STKGPSVFPL APSSKSTSGG M2_33H IG............YFDYWG QGTLVTVSSA STKGPSVFPL APSSKSTSGG M2_34H IG........ ....YFDYWGQGTLVTVSSA STKGPSVFPL APSSKSTSGG M2_35H IG........ ....YFDYWG QGTLVTVSSASTKGPSVFPL APSSKSTSGG151                                                200 M2_11H TAALGCLVKDYFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV M2_12H TAALGCLVKD YFPEPVTVSWNSGALTSGVH TFPAVLQSSG LYSLSSVVTV M2_16H TAALGCLVKD YFPEPVTVSW NSGALTSGVHTFPAVLQSSG LYSLSSVVTV M2_18H TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSGLYSLSSVVTV M2_20H TAALGCLVKD YFPEPVTVSW KSGALTSGVH TFPAVLQSSG LYSLSSVVTVM2_31H TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV M2_32HTAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV M2_33H TAALGCLVKDYFPEPVTVSW NSGALTSGVH TFPAVLQSSG LYSLSSVVTV M2_34H TAALGCLVKD YFPEPVTVSWNSGALTSGVH TFPAVLQSSG LYSLSSVVTV M2_35H TAALGCLVKD YFPEPVTVSW NSGALTSGVHTFPAVLQSSG LYSLSSVVTV 201                                  237 M2_11HPSSSLGTQTY ICNVNHKPSN TKVDKKAEPK SHHHHHH M2_12H PSSSLGTQTY ICNVNHKPSSTKVDKKAEPK SHHHHHH M2_16H PSSSLGTQTY ICNVNHKPSN TKVDKKAEPK SHHHHHHM2_18H PSSSLGTQTY ICNVNHKPSN TKVDKKAEPK SHHHHHH M2_20H PSSSLGTQTYICNVNHKPSN TKVDKKAEPK SHHHHHH M2_31H PSSSLGTQTY ICNVNHKPSN TKVDKKAEPKSHHHHHH M2_32H PSSSLGTQTY ICNVNHKPSN TKVDKKAEPK SHHHHHH M2_33HPSSSLGTQTY ICNVNHKPSN TKVDKKAEPK SHHHHHH M2_34H PSSSLGTQTY ICNVNHKPSNTKYDKKAEPK SHHHHHH M2_35H PSSSLGTQTY ICNVNHKPSN TKVDKKAEPK SHHHHHH M2-LKappa Chain VKCK 10-10M Affinity Cut (Thu Sep 23) (SEQ ID NOS: 72, 74,76, 78, 80, 82, 84, 86, 88, and 90 respectively)1                                                   50 M2_11L EIVMTQSPGTLSLSPGERAT LSCRASQGVS SSYLAWYQQK PGQAPRLLIY M2_12L EIVMTQSPGT LSLSPGERATLSCRASQGVS SSYLAWYQQK PGQAPRLLIY M2_16L EIVMTQSPGT LSLSPGERAT LSCRASQSVSSSYLAWYQQK PGQAPRLLIY M2_18L EIVMTQSPGT LSLSPGERAT LSCRASQSVS STYLAWYQQKPGQAPRLLIY M2_20L EIVMTQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIYM2_31L EIVLTQSPAT LSLSPGERAT LSCRASQSVS S.YLAWYQQK PGQAPRLLIY M2_32LEIVLTQSPAT LSLSPGERAT LSCRASQSVS S.YLAWYQQK PGQAPRLLIY M2_33L EIVLTQSPGTLSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY M2_34L EIVLTQSPAT LSLSPGERATLSCRASQSVS S.YLAWYQQK PGQAPRLLIY M2_35L EIVLTQSPAT LSLSPGERAT LSCRASQSVSS.YLAWYQQK PGQAPRLLIY51                                                 100 M2_11L GASSRATGIPDRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYGSSPPFTF M2_12L GASSRATGIP DRFSGSGSGTDFTLTISSLE PEDFAVYYCQ QYGSSPPYTF M2_16L GASSRATGIP DRFSVSGSGT DFTLTISRLEPEDFAVYYCQ QYGSS..FTF M2_18L GASSRATGTP DRFSGSGSGT DFTLTISRLE PEDFAVYYCQQYVSS..FTF M2_20L GASRRATGIP DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYGSSPMYTFM2_31L DASNRATGIP ARFSGSGSGT DFTLTISSLE PEDFAVYYCQ QRTNWP.RTF M2_32LDASNRAAGIP ARFSGSGSGT DFTLTISSLE PEDFAVYYCQ QRNNWP.LTF M2_33L GASSRATGIPDRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYGSSPPYTF M2_34L DASNRATGIP ARFSGSGSGTDFTLTTSSLE PEDFAVYYCQ QRTNWP.RTF M2_35L DASNRATGIP ARFSGSGSGT DFTLTISSLEPEDFAVYYCQ QRTNWP.RTF101                                                150 M2_11L GPGTKVDIKRTVAAPSVFIF PPSDEQLRSG TASVVCLLNN FYPREAKVQW M2_12L GQGTKLEIKR TVAAPSVFIFPPSDEQLKSG TASVVCLLNN FYPREAKVQW M2_16L GPGTKVDIKR TVAAPSVFIF PPSDEQLKSGTASVVCLLNN FYPREAKVQW M2_18L GPGTKVDIKR TVAAPSVFIF PPSDEQLKSG TASVVCLLNNFYPREAKVQW M2_20L GQGTKLEIKR TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQWM2_31L GQGTKVEIKR TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW M2_32LGGGTKVEIKR TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW M2_33L GQGTKLEIKRTVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW M2_34L GQGTKVEIKR TVAAPSVFIFPPSDEQLKSG TASVVCLLNN FYPREAKVQW M2_35L GQGTKVEIKR TVAAPSVFIF PPSDEQLKSGTASVVCLLNN FYPREAKVQW151                                                200 M2_11L KVDNALQSGNSQESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH M2_12L KVDNALQSGN SQESVTEQDSKDSTYSLSST LTLSKADYEK HKVYACEVTH M2_16L KVDNALQSGN SQESVTEQDS KDSTYSLSSTLTLSKADYEK HKVYACEVTH M2_18L KVDNALQSGN SQESVTEQDS KDSTYSLSST LTLSKADYEKHKVYACEVTH M2_20L KVDNALQSGN SQESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTHM2_31L KVDNALQSGN SQESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH M2_32LKVDNALQSGN SQESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH M2_33L KVDNALQSGNSQESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH M2_34L KVDNALQSGN SQESVTEQDSKDSTYSLSST LTLSKADYEK HKVYACEVTH M2_35L KVDNALQSGN SQESVTEQDS KDSTYSLSSTLTLSKADYEK HKVYACEVTH 201                      226 M2_11L QGLSSPVTKSFNRGESYPYD VPDYAS M2_12L QGLSSPVTKS FNRGESYPYD VPDYAS M2_16L QGLSSPVTKSFNRGESYPYD VPDYAS M2_18L QGLSSPVTKS FNRGESYPYD VPDYAS M2_20L QGLSSPVTKSFNRGESYPYD VPDYAS M2_31L QGLSSPVTKS FNRGESYPYD VPDYAS M2_32L QGLSSPVTKSFNRGESYPYD VPDYAS M2_33L QGLSSPVTKS FNRGESYPYD VPDYAS M2_34L QGLSSPVTKSFNRGESYPYD VPDYAS M2_35L QGLSSPVTKS FNRGESYPYD VPDYAS

EXAMPLE 23 Growth of E. coli Cultures and Purification of RecombinantAntibodies and Antigens

A shake flask inoculum is generated overnight from a −70° C. cell bankin an Innova 4330 incubator shaker (New Brunswick Scientific, Edison,N.J.) set at 37° C., 300 rpm. The inoculum is used to seed a 20 μLfermenter (Applikon, Foster City, Calif.) containing defined culturemedium (Pack, et al., Bio/Technology 11:1271-1277 (1993)) supplementedwith 3 μg/L L-leucine, 3 μg/L L-isoleucine, 12 μg/L casein digest(Difco, Detroit, Mich.), 12.5 μg/L glycerol and 10 mg/ml tetracycline.The temperature, pH and dissolved oxygen in the fermenter are controlledat 26° C., 6.0-6.8 and 25% saturation, respectively. Foam is controlledby addition of polypropylene glycol (Dow, Midland, Mich.). Glycerol isadded to the fermenter in a fed-batch mode. Fab expression is induced byaddition of L(+)-arabinose (Sigma, St. Louis, Mo.) to 2 μg/L during thelate logarithmic growth phase. Cell density is measured by opticaldensity at 600 nm in an UV-1201 spectrophotometer (Shimadzu, Columbia,Md.). Final Fab concentrations are typically 100-500 mg/L. Following runtermination and adjustment of pH to 6.0, the culture is passed twicethrough an M-210B-EH Microfluidizer (Microfluidics, Newton, Mass.) at17000 psi. The high pressure homogenization of the cells releases theFab into the culture supernatant.

The first step in purification is expanded bed immobilized metalaffinity chromatography (EB-IMAC). Streamline Chelating resin(Pharmacia, Piscataway, N.J.) is charged with 0.1 M NiCl₂. It is thenexpanded and equilibrated in 50 mM acetate, 200 mM NaCl, 10 mMimidazole, 0.01% NaN₃, pH 6.0 buffer flowing in the upward direction. Astock solution is used to bring the culture homogenate to 10 mMimidazole, following which, it is diluted two-fold or higher inequilibration buffer to reduce the wet solids content to less than 5% byweight. It is then loaded onto the Streamline column flowing in theupward direction at a superficial velocity of 300 cm/hr. The cell debrispasses through unhindered, but the Fab is captured by means of the highaffinity interaction between nickel and the hexahistidine tag on the Fabheavy chain. After washing, the expanded bed is converted to a packedbed and the Fab is eluted with 20 mM borate, 150 mM NaCl, 200 mMimidazole, 0.01% NaN₃, pH 8.0 buffer flowing in the downward direction.The second step in purification uses ion-exchange chromatography (IEC).Q Sepharose FastFlow resin (Pharmacia, Piscataway, N.J.) is equilibratedin 20 mM borate, 37.5 mM NaCl, 0.01% NaN₃, pH 8.0. The Fab elution poolfrom the EB-IMAC step is diluted four-fold in 20 mM borate, 0.01% NaN₃,pH 8.0 and loaded onto the IEC column. After washing, the Fab is elutedwith a 37.5-200 mM NaCl salt gradient. The elution fractions areevaluated for purity using an Xcell II SDS-PAGE system (Novex, SanDiego, Calif.) prior to pooling. Finally, the Fab pool is concentratedand diafiltered into 20 mM borate, 150 mM NaCl, 0.01% NaN₃, pH 8.0buffer for storage. This is achieved in a Sartocon Slice system fittedwith a 10,000 MWCO cassette (Sartorius, Bohemia, N.Y.). The finalpurification yields are typically 50%. The concentration of the purifiedFab is measured by UV absorbance at 280 nm, assuming an absorbance of1.6 for a 1 mg/mL solution.

EXAMPLE 24 Generation of Cmu Targeted Mice

The following example describes the making of mice with disrupted, andthus non-functional, imnmunoglobulin genes.

Construction of a CMD Targeting Vector

To disrupt the mouse immunoglobulin gene, a vector containing a fragmentof a murine Ig heavy chain locus is transfected into a mouse embryoniccell. The mouse Ig heavy chain sequence “targets” the vector to themouse immunoglobulin gene locus. The following describes construction ofthis immunoglobulin gene “targeting” vector.

The plasmid pICEmu contains an EcoRI/XhoI fragment of the murine Igheavy chain locus, spanning the mu gene, that was obtained from a Balb/Cgenomic lambda phage library (Marcu et al. Cell 22: 187, 1980). Thisgenomic fragment was subcloned into the XhoI/EcoRI sites of the plasmidpICEMI9H (Marsh et al; Gene 32, 481-485, 1984). The heavy chainsequences included in pICEmu extend downstream of the EcoRI site locatedjust 3′ of the mu intronic enhancer, to the XhoI site locatedapproximately 1 kb downstream of the last transmembrane exon of the mugene; however, much of the mu switch repeat region has been deleted bypassage in E. coli.

The targeting vector was constructed as follows (See FIG. 6). A 1.3 kbHindIII/SmaI fragment was excised from pICEmu and subcloned intoHindIII/SmaI digested pBluescript (Stratagene, La Jolla, Calif.). ThispICEmu fragment extends from the HindIII site located approximately 1 kb5′ of Cmu1 to the SmaI site located within Cmu1. The resulting plasmidwas digested with SmaI/SpeI and the approximately 4 kb SmaI/XbaIfragment from pICEmu, extending from the Sma I site in Cmu1 3′ to theXbaI site located just downstream of the last Cmu exon, was inserted.

The resulting plasmid, pTAR1, was linearized at the SmaI site, and a neoexpression cassette inserted. This cassette consists of the neo geneunder the transcriptional control of the mouse phosphoglycerate kinase(pgk) promoter (XbaI/TaqI fragment; Adra et al. (1987) Gene 60: 65-74)and containing the pgk polyadenylation site (PvuII/HindIII fragment;Boer et al. (1990) Biochemical Genetics 28: 299-308). This cassette wasobtained from the plasmid pKJ1 (described by Tybulewicz et al. (1991)Cell 65: 1153-1163) from which the neo cassette was excised as anEcoRI/HindIII fragment and subcloned into EcoRI/HindIII digestedpGEM-7Zf (+) to generate pGEM-7 (KJ1). The neo cassette was excised frompGEM-7 (KJ1) by EcoRI/SalI digestion, blunt ended and subcloned into theSmaI site of the plasmid pTAR1, in the opposite orientation of thegenomic Cmu sequences.

The resulting plasmid was linearized with Not I, and a herpes simplexvirus thymidine kinase (tk) cassette was inserted to allow forenrichment of ES clones (mouse embryo-derived stem cells) bearinghomologous recombinants, as described by Mansour et al. (1988) Nature336: 348-352. This cassette consists of the coding sequences of the tkgene bracketed by the mouse pgk promoter and polyadenylation site, asdescribed by Tybulewicz et al. (1991) Cell 65: 1153-1163. The resultingCMD targeting vector contains a total of approximately 5.3 kb ofhomology to the heavy chain locus and is designed to generate a mutantmu gene into which has been inserted a neo expression cassette in theunique SmaI site of the first Cmu exon. The targeting vector waslinearized with PvuI, which cuts within plasmid sequences, prior toelectroporation into ES cells.

Generation and Analysis of Targeted ES Cells.

The vector containing the murine Ig heavy chain gene fragment is theninserted into a mouse embryonic stem cell (an ES cell). The followingdescribes the construction of this immunoglobulin gene-containing vector“targeted” ES cell and analysis of the ES cells' DNA after the vectorhas been inserted (i.e., transfected) into the cell.

AB-1 ES cells (McMahon, A. P. and Bradley, A., (1990) Cell 62:1073-1085) were grown on mitotically inactive SNL76/7 cell feeder layers(ibid.) essentially as described (Robertson, E. J. (1987) inTeratocarcinomas and Embryonic Stem Cells: a Practical Approach (E. J.Robertson, ed.) Oxford: IRL Press, p. 71-112). The linearized CMDtargeting vector was electroporated into AB-1 cells by the methodsdescribed Hasty et al. (Hasty, P. R. et al. (1991) Nature 350: 243-246).Electroporated cells were plated into 100 mm dishes at a density of1-2×106 cells/dish. After 24 hours, G418 (200 micrograms/ml of activecomponent) and FIAU (5×10−7 M) were added to the medium, anddrug-resistant clones were allowed to develop over 8-9 days. Clones werepicked, trypsinized, divided into two portions, and further expanded.Half of the cells derived from each clone were then frozen and the otherhalf analyzed for homologous recombination between vector and targetsequences.

DNA analysis was carried out by Southern blot hybridization. DNA wasisolated from the clones as described Laird et al. (Laird, P. W. et al.,(1991) Nucleic Acids Res. 19: 4293). Isolated genomic DNA was digestedwith SpeI and probed with a 915 bp SacI fragment, probe A (FIG. 6),which hybridizes to a sequence between the mu intronic enhancer and themu switch region. Probe A detects a 9.9 kb SpeI fragment from the wildtype locus, and a diagnostic 7.6 kb band from a mu locus which hashomologously recombined with the CMD targeting vector (the neoexpression cassette contains a SpeI site). Of 1132 G418 and FIAUresistant clones screened by Southern blot analysis, 3 displayed the 7.6kb Spe I band indicative of homologous recombination at the mu locus.These 3 clones were further digested with the enzymes BglI, BstXI, andEcoRI to verify that the vector integrated homologously into the mugene. When hybridized with probe A, Southern blots of wild type DNAdigested with BglI, BstXI, or EcoRI produce fragments of 15.7, 7.3, and12.5 kb, respectively, whereas the presence of a targeted mu allele isindicated by fragments of 7.7, 6.6, and 14.3 kb, respectively. All 3positive clones detected by the SpeI digest showed the expected BglI,BstXI, and EcoRI restriction fragments diagnostic of insertion of theneo cassette into the Cmu1 exon.

Generation of Mice Bearing the Mutated Mu Gene

The three targeted ES clones, designated number 264, 272, and 408, werethawed and injected into C57BL/6J blastocysts as described by Bradley(Bradley, A. (1987) in Teratocarcinomas and Embryonic Stem Cells: aPractical Approach. (E. J. Robertson, ed.) Oxford: IRL Press, p.113-151). Injected blastocysts were transferred into the uteri ofpseudopregnant females to generate chimeric mice representing a mixtureof cells derived from the input ES cells and the host blastocyst. Theextent of ES cell contribution to the chimera can be visually estimatedby the amount of agouti coat coloration, derived from the ES cell line,on the black C57BL/6J background. Clones 272 and 408 produced only lowpercentage chimeras (i.e. low percentage of agouti pigmentation) butclone 264 produced high percentage male chimeras. These chimeras werebred with C57BL/6J females and agouti offspring were generated,indicative of germline transmission of the ES cell genome. Screening forthe targeted mu gene was carried out by Southern blot analysis of BglIdigested DNA from tail biopsies (as described above for analysis of EScell DNA). Approximately 50% of the agouti offspring showed ahybridizing BglI band of 7.7 kb in addition to the wild type band of15.7 kb, demonstrating a germline transmission of the targeted mu gene.

Analysis of Transgenic Mice for Functional Inactivation of Mu Gene.

To determine whether the insertion of the neo cassette (including the Igheavy chain sequence) into Cmu1 has inactivated the Ig heavy chain gene,a clone 264 chimera was bred with a mouse homozygous for the JHDmutation, which inactivates heavy chain expression as a result ofdeletion of the JH gene segments (Chen et al, (1993) Immunol. 5:647-656). Four agouti offspring were generated. Serum was obtained fromthese animals at the age of 1 month and assayed by ELISA for thepresence of murine IgM. Two of the four offspring were completelylacking IgM (Table 2). Genotyping of the four animals by Southern blotanalysis of DNA from tail biopsies by BglI digestion and hybridizationwith probe A (FIG. 6), and by StuI digestion and hybridization with a475 bp EcoRI/StuI fragment (ibid.) demonstrated that the animals whichfail to express serum IgM are those in which one allele of the heavychain locus carries the JHD mutation, the other allele the Cmu1mutation. Mice heterozygous for the JHD mutation display wild typelevels of serum Ig. These data demonstrate that the Cmu1 mutationinactivates expression of the mu gene.

TABLE 2 Level of serum IgM, detected by ELISA, for mice carrying boththe CMD and JHD mutations (CMD/JHD), for mice heterozygous for the JHDmutation (+/JHD), for wild type (129 Sv × C57BL/6J)F1 mice (+/+), andfor B cell deficient mice homozygous for the JHD mutation (JHD/JHD).Mouse Serum IgM (micrograms/ml) Ig H chain genotype 42 <0.002 CMD/JHD 43196 +/JHD 44 <0.002 CMD/JHD 45 174 +/JHD 129 × BL6 F1 153 +/+ JHD <0.002JHD/JHD

EXAMPLE 25 Generation of HCo12 Transgenic Mice

The following describes the generation of transgenic mice containinghuman immunoglobulin heavy chain gene sequence that can generate humanimmunoglobulins. Because these mice cannot make endogenous (i.e., mouse)immunoglobulins, upon challenge with antigen, e.g., a human polypeptide,only human sequence immunoglobulins are made by the transgenic mouse.

The HCo12 Human Heavy Chain Transgene.

The HCo12 transgene was generated by coinjection of the 80 kb insert ofpHC2 (Taylor et al., 1994, Int. Immunol., 6: 579-591) and the 25 kbinsert of pVx6. The plasmid pVx6 was constructed as described below. An8.5 kb HindIII/SalI DNA fragment, comprising the germline human VH1-18(DP-14) gene together with approximately 2.5 kb of 5′ flanking, and 5 kbof 3′ flanking genomic sequence was subcloned into the plasmid vectorpSP72 (Promega, Madison, Wis.) to generate the plasmid p343.7.16. A 7 kbBamHI/HindIII DNA fragment, comprising the germline human VH5-51 (DP-73)gene together with approximately 5 kb of 5′ flanking and 1 kb of 3′flanking genomic sequence, was cloned into the pBR322 based plasmidcloning vector pGP1f (Taylor et al. 1992, Nucleic Acids Res. 20:6287-6295), to generate the plasmid p251f.

A new cloning vector derived from pGP1f, pGP1k (Seq. ID #1), wasdigested with EcoRV/BamHI, and ligated to a 10 kb EcoRV/BamHI DNAfragment, comprising the germline human VH3-23 (DP47) gene together withapproximately 4 kb of 5′ flanking and 5 kb of 3′ flanking genomicsequence. The resulting plasmid, p112.2RR.7, was digested withBamHI/SalI and ligated with the 7 kb purified BamHI/SalI insert ofp251f. The resulting plasmid, pVx4, was digested with XhoI and ligatedwith the 8.5 kb XhoI/SalI insert of p343.7.16.

A plasmid clone was obtained with the V_(H)1-18 gene in the sameorientation as the other two V genes. This clone, designated pVx6, wasthen digested with NotI and the purified 26 kb insertcoinjected—together with the purified 80 kb NotI insert of pHC2 at a 1:1molar ratio—into the pronuclei of one-half day (C57BL/6J x DBA/2J)F2embryos as described by Hogan et al. (B. Hogan et al., Manipulating theMouse Embryo, A Laboratory Manual, 2nd edition, 1994, Cold Spring HarborLaboratory Press, Plainview N.Y.).

Three independent lines of transgenic mice comprising sequences fromboth Vx6 and HC2 were established from mice that developed from theinjected embryos. These lines of transgenic mice are designated(HCo12)14881, (HCo12)15083, and (HCo12)15087. Each of the three lineswere then bred with mice comprising the CMD mutation described inExample 23, the JKD mutation (Chen et al. 1993, EMBO J. 12: 811-820),and the (KCo5)9272 transgene (Fishwild et al. 1996, Nature Biotechnology14: 845-851). The resulting mice express human heavy and kappa lightchain transgenes (and produce human sequence heavy and kappa light chainantibodies) in a background homozygous for disruption of the endogenousmouse heavy and kappa light chain loci.

Two different strains of mice were used to generate hybridomas andmonoclonal antibodies reactive to human IL-8. Strain ((CMD)++; (JKD)++;(HCo7)11952+/++; (KCo5)9272+/++), and strain ((CMD)++; (JKD)++;(HCo12)15087+/++; (KCo5)9272+/++). Each of these strains are homozygousfor disruptions of the endogenous heavy chain (CMD) and kappa lightchain (JKD) loci. Both strains also comprise a human kappa light chaintransgene (HCo7), with individual animals either hemizygous orhomozygous for insertion #11952. The two strains differ in the humanheavy chain transgene used. Mice were hemizygous or homozygous foreither the HCo7 or the HCo12 transgene. The CMD mutation is describedabove in Example 23, above. The generation of(HCo12)15087 mice isdescribed above. The JKD mutation (Chen et al. 1993, EMBO J. 12:811-820) and the (KCo5)9272 (Fishwild et al. 1996, Nature Biotechnology14: 845-851) and (HCo7)11952 mice, are described in U.S. Pat. No.5,770,429 (Lonberg & Kay, Jun. 23, 1998).

EXAMPLE 26 Preparation of Biotinylated Antibodies

Purified antibodies were dialyzed against a minimum of 100 volumes ofphosphate buffered saline (PBS), pH 7.4, for at least 4 hours.Antibodies were diluted to a final concentration of 2 mg/ml in PBS. Astock solution containing 40 mM of biotin-XX-NHS ester (MolecularProbes, Eugene, Oreg.) was prepared in dimethylsulfoxide. Thebiotin-XX-NHS solution was added to antibodies at a final concentrationof 0.4 mM and reacted for 90 minutes at room temperature.Aminoethanesulfonic acid was added to a final concentration of 20 mM andincubated for five minutes to quench remaining reactive groups. Thebiotinylated antibodies were dialyzed extensively to remove smallmolecules containing biotin from the antibodies.

Preparation of Alkaline Phosphatase-antibody Conjugates

Alkaline phosphatase (AP, Calzyme Laboratories, San Luis Obispo, Calif.)was placed into dialysis using a minimum of 100 volumes of column buffer(50 mM potassium phosphate, 10 mM borate, 150 mM NaCl, 1 mM MgSO₄, pH7.0) at 2-8° C. for at least four hours. The buffer was changed at leasttwice prior to use of the AP. When the AP was removed from dialysis andbrought to room temperature, the concentration was determined byabsorbance at 280 nm using an absorbance of 0.77 for a 1 mg/mL solution.The AP was diluted to 5 mg/mL with column buffer. The reaction of AP andsuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC,Pierce Chemical Co., Rockford, Ill.) was carried out using a 20:1 ratioof SMCC:AP. SMCC was dissolved in acetonitrile at 20 mg/mL and dilutedby a factor of 84 when added to AP while vortexing or rapidly stirring.The solution was allowed to stand at room temperature for 90 minutesbefore the unreacted SMCC and low molecular weight reaction productswere separated from the AP using gel filtration chromatography (G50Fine, Pharmacia Biotech, Piscataway, N.J.) in a column equilibrated withcolumn buffer.

Antibodies were dialyzed in a minimum of 100 volumes of PBS and dilutedto 2 mg/mL in PBS. A solution containing 40 mM N-succinimidylS-acetylthiopropionate (SATP, Pierce Chemical Co., Rockford, Ill.) wasprepared in dimethylsulfoxide, diluted to a final concentration of 0.32mM in the antibody solution, and incubated for 90 minutes at roomtemperature. Aminoethanesulfonic acid was added to a final concentrationof 20 mM and incubated for 5 minutes to stop the reaction. The modifiedantibody was dialyzed using a minimum of 100 volumes of 50 mM potassiumphosphate, 10 mM borate, 150 mM NaCl, pH 7.0, for a minimum of 4 hoursbefore the dialysis buffer was changed and dialysis continued for atleast four hours. The antibody concentration was determined using theabsorbance at 280 nm with an absorbance of 1.6 corresponding to a 1mg/mL solution. Hydroxylamine was dissolved to a concentration of 0.5 Min 0.1 M potassium phosphate, 10 mM ethylenediaminetetraacetic acid, pH7.2. This solution was diluted into the antibody solution to achieve afinal concentration of hydroxylamine of 50 mM and incubated for 2 hoursat room temperature. Modified antibody was mixed with SMCC-AP inequimolar amounts and incubated for one hour at room temperature beforeadding β-mercaptoethanol to a final concentration of 1 mM, incubatingfor 5 minutes, adding N-ethylmaleimide to a final concentration of 2 mMand incubating for 5 minutes. Antibody-enzyme conjugates were separatedfrom unconjugated antibody by gel filtration chromatography usingSEPHACRYL™ S-200 (Pharmacia Biotech, Piscataway, N.J.) in column buffer.Conjugates were diluted into block solution for use in immunoassays.

EXAMPLE 27 Epitope Mapping of Human Monoclonal Antibodies to IL-8

A BIACORE® 3000 instrument (Biacore AB, Uppsala, Sweden) was used tomeasure the epitope binding. Goat anti-human kappa antibody (FisherScientific, Pittsburgh, Pa.) was immobilized onto a CM5 sensor chip(Biacore AB, Uppsala, Sweden) as described in Biacore Application note101 (Biacore AB, Uppsala, Sweden), except goat anti-human kappa antibodywas diluted into 10 mM sodium acetate, pH 4.0.

Fourteen human monoclonal antibodies (Example 15) were used to map theepitopes of IL-8 using a two-site assay (Johne et al., Journal ofImmunological Methods, 160 (1993) 191-198). Each antibody was diluted to100 μg/mL and the IL-8 was diluted to 1 μM in 10 mMN-2-hydroxyethylpiperazine-N′-2-ethane-sulfonic acid, 150 mM sodiumchloride, 3 mM EDTA, 0.005% polysorbate 20, pH7.4 (HBS-EP, Biacore AB,Uppsala, Sweden). The primary antibody (M1-10) was bound to the goatanti-human kappa antibody on the CM5 chip by injecting 20 μL of antibodyover all 4 channels. IL-8 (10 μL) was then bound to the primary antibodyin all 4 channels. One secondary antibody (10 μL) was passed through onechannel each. The resonance units of the secondary antibodies weremeasured 20 seconds after the injection was over. Blank resonance unitvalues for each primary/secondary antibody combination were obtained byinjecting HBS-EP instead of IL-8 and these were subtracted from theresonance units measured for the corresponding primary/secondaryantibody pairing that was contacted with IL-8. Blocking antibodyinjections were not done. The high levels of resonance units for allantibody combinations except M1-10 with itself indicate that all of theantibodies listed except M1-10 bind to an epitope that is different fromthe epitope bound by M1-10 and that the binding of these antibodies islargely unaffected by the binding of M1-10 to IL-8. Data not shownindicate that the binding to IL-8 of all antibodies listed other thanM1-10 is substantially affected by the prior binding of any one of themto IL-8.

Secondary Monoclonal Antibody Resonance Units M1-3 839 M1-4 911 M1-5 820M1-8 888 M1-10 −17 M1-21 706 M1-23 796 M1-25 925 M2-11 798 M2-12 794M2-16 818 M2-18 754 M2-20 848 M2-34 799

EXAMPLE 28 Experimental Procedures

A sequential sandwich enzyme-linked immunosorbent assay (ELISA) wasperformed to create a standard curve of response versus concentrationand to determine the assay sensitivity for each antibody set. All assayswere performed in 384-well black polystyrene streptavidin-coatedmicroplates (Pierce Chemical Co., Rockford, Ill.) with an averagebinding capacity of approximately 50 femtomoles biotin/well. Alkalinephosphatase (AP)-labeled antibodies were used to bind antigen capturedby antibody on the solid phase, and the binding was detected usingAttoPhos substrate (JBL Scientific, San Luis Obispo, Calif.). Theantibody sets that were analyzed were polyclonal murineantibody-biotin/polyclonal murine antibody-AP, polyclonal humanantibody-biotin/polyclonal human antibody-AP, and monoclonal humanantibodyM1-10-biotin/monoclonal human antibody M1-25-AP. Both polyclonalhuman and polyclonal murine antibodies were selected to bind IL-8 usinga lower affinity cutoff of 10⁹ M⁻¹. Both monoclonal antibodies wereselected from the polyclonal human library. The polyclonal mouseantibody library was obtained as described in PCT 98/06704, filed, Apr.3, 1998 using multiple rounds of selection with biotinylated IL-8 at 1nM. Reagent pipetting was performed using a TECAN Genesis RSP 200/8robotic sample processor (TECAN U.S., Inc., Research Triangle Park,N.C.). Individual microplate wells were washed by a TECAN Columbus384-well strip washer, and kinetic fluorescence in each microplate wellwas determined by a TECAN SpectraFluor Plus microplate reader using anexcitation wavelength of 430 nm and an emission wavelength of 570 nm.All pipetting methods were programmed using TECAN Gemini 3.0 liquidhandling software. All TECAN robotic resources were controlled using theTECAN multischeduler software FACTS 4.5.

Samples to be analyzed were diluted 1:4.5 in conjugate diluent (CD8; 10mM Tris, 150 mM sodium chloride, 1 mM magnesium chloride, 0.1 mM zincchloride, 0.1% polyvinyl alcohol, 1% bovine serum albumin, 0.1% sodiumazide, at pH 8.15). Standard curves were created using samples of eitherconjugate diluent or pooled normal plasma with various concentrations ofinterleukin-8 (IL-8) added. 40 μl of 2 μg/ml biotinylated polyclonalmurine antibody, polyclonal human antibody or monoclonal human antibodyM1-10 in CD8 were pipetted into individual microplate wells. Themicroplate was incubated for 1 hour at 25° C. and individual wells werewashed three times with wash buffer (20 mM borate, 150 mM sodiumchloride, 0.02% polyoxyethylene 20-sorbitan monolaurate, 0.1% sodiumazide, at pH 8.2) in overflow mode. 40 μl of the sample to be analyzedwas pipetted into individual microplate wells, and the plate wasincubated for 1 hour at 25° C. Individual microplate wells were washedthree times with wash buffer, and 40 μl of 5 μg/ml alkalinephosphatase-labeled antibody in CD8 was pipetted into individualmicroplate wells. Polyclonal murine antibody-AP, polyclonal humanantibody-AP, and monoclonal human antibody M1-25-AP were paired withpolyclonal murine antibody-biotin, polyclonal human antibody-biotin, andmonoclonal human antibody M1-10-biotin, respectively. The microplate wasincubated for 1 hour at 25° C. and individual wells were washed sixtimes with wash buffer. 40 μl of 1 mM AttoPhos substrate in 2.4 Mdiethanolamine, 0.057 mM magnesium chloride, 0.005% sodium azide at pH10.0 was pipetted into each microplate well, and the kineticfluorescence was read for 20 minutes. Samples known to containhuman-anti-mouse antibodies (HAMA) or heterophilic antibodies werepurchased (Scantibodies, Inc., Santee, Calif.). All HAMA-positive andstandard curve samples were analyzed in quadruplicate.

All statistical calculations and graphs used for data analysis wereprepared using Microsoft Excel 97. The kinetic fluorescence of each wellwas quantified by calculating the slope of the response, in units of0.1×milli-relative fluorescent units per second (0.1×mRFU/sec). Astandard curve was created for each antibody set by plottingblank-corrected slope (0.1×mRFU/sec; y-axis) versus concentration(x-axis). The correlation coefficient for the linear portion of thecurve was determined. Assay sensitivity was calculated from the standardcurve and is defined as the analyte concentration corresponding to aslope equal to the slope plus two standard deviations of the assay blank(negative control; no analyte present). Slope (0.1×mRFU/sec) wasconverted to concentration (pM) for all HAMA-positive samples using thestandard curve created from plasma samples with various concentrationsof IL-8 added.

The sensitivity of the polyclonal murine antibody, polyclonal humanantibody for detecting the IL-8 concentration in human plasma samplesusing a sequential sandwich ELISA was calculated to be 65.2 pM, 30.7 pM,and 1.3 pM, respectively. The correlation coefficients for theblank-corrected linear portion of the standard curve of the polyclonalmurine antibody, polyclonal human antibody for detecting the IL-8concentration in human plasma samples using a sequential sandwich ELISAwas calculated to be 0.912, 0.993, and 0.998, respectively.

The data provided in the Table show a dramatic reduction in the valuesfor IL-8 concentration as a result of using human antibodies. Becauseboth human antibody-based assays are substantially more sensitive thanthe murine antibody-based assay, these results indicate that theapparent IL-8 concentrations determined in these samples using thepolyclonal murine antibody-based assay that are above the sensitivitylimit of that assay are falsely elevated due to HAMA or heterophilicantibodies in the samples.

TABLE 5 SLOPE (0.1 × mRFU/sec), BLANK CORRECTED [IL-8] (pM) MONOCLONALMONOCLONAL POLY- POLY- HUMAN HUMAN CLONAL CLONAL ANTIBODIES POLYCLONALPOLYCLONAL ANTIBODIES MURINE HUMAN M1 MURINE HUMAN M1 SAMPLE ID ANTIBODYANTIBODY 10/M1-25 ANTIBODY ANTIBODY 10/M1-25 HAMA-POSITIVE HUMAN PLASMA11882-201 0.2671 0.5830 0.6530 31.42 1.27 1.30 11879-966 0.4351 0.22600.1397 51.19 0.49 0.28  2172-51 4.3512 3.9161 3.7688 511.91 8.55 7.4911658-332 16.0843 0.9385 1.7111 1892.27 2.05 3.40 11879-857 46.82860.8811 0.3661 5509.25 1.92 0.73  2161-17 20.1484 0.7397 0.9231 2370.401.61 1.84 11707-22 7.4092 1.7971 2.5912 871.67 3.92 5.15 11707-3116.0596 0.1015 2.4966 1889.36 0.22 4.96 11879-819 5.1529 0.3544 0.5127606.22 0.77 1.02 11658-88 2.3892 0.6264 0.7798 281.08 1.37 1.55  2160-520.8846 0.6428 1.0283 104.07 1.40 2.04  2154-7 25.5515 0.8860 2.76643006.06 1.93 5.50 10132-523 54.9596 0.6422 0.6183 6465.83 1.40 1.23 9881-276 6.9326 1.6051 2.2132 815.60 3.50 4.40 11906-47 5.5530 0.27340.7166 653.29 0.60 1.43 11879-210 50.7084 0.7032 0.5550 5965.69 1.541.10 HETEROPHILIC HUMAN PLASMA 10049-320 8.4718 0.3944 −0.1345 996.680.86 −0.27  2217-1 0.1872 0.1965 0.1952 22.03 0.43 0.39 10049-114 0.54700.5707 0.1850 64.36 1.25 0.37 10060-285 0.4577 0.2119 0.0569 53.84 0.460.11

SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 110 <210> SEQ ID NO 1 <211>LENGTH: 43 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence:Oligo 188 <400> SEQUENCE: 1 ttacccctgt ggcaaaagcc gaagtgcagctggtggagtc tgg 43 <210> SEQ ID NO 2 <211> LENGTH: 43 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence:Oligo 944 <400>SEQUENCE: 2 ttacccctgt ggcaaaagcc caggtgcagc tggtgcagtc tgg 43 <210> SEQID NO 3 <211> LENGTH: 43 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence:Oligo 948 <400> SEQUENCE: 3 ttacccctgt ggcaaaagcccaggtgcagc tggtggagtc tgg 43 <210> SEQ ID NO 4 <211> LENGTH: 20 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence:Oligo 952 <400>SEQUENCE: 4 gatgggccct tggtggaggc 20 <210> SEQ ID NO 5 <211> LENGTH: 43<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence:Oligo 189 <400>SEQUENCE: 5 ctgcccaacc agccatggcc gaaattgtgc tcacccagtc tcc 43 <210> SEQID NO 6 <211> LENGTH: 46 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence:Oligo 931 <400> SEQUENCE: 6 tcgctgccca accagccatggccgtcatct ggatgaccca gtctcc 46 <210> SEQ ID NO 7 <211> LENGTH: 46 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence:Oligo 932 <400>SEQUENCE: 7 tcgctgccca accagccatg gccaacatcc agatgaccca gtctcc 46 <210>SEQ ID NO 8 <211> LENGTH: 46 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence:Oligo 933 <400> SEQUENCE: 8 tcgctgccca accagccatggccgccatcc ggatgaccca gtctcc 46 <210> SEQ ID NO 9 <211> LENGTH: 46 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence:Oligo 934 <400>SEQUENCE: 9 tcgctgccca accagccatg gccgccatcc agttgaccca gtctcc 46 <210>SEQ ID NO 10 <211> LENGTH: 46 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence:Oligo 935 <400> SEQUENCE: 10 tcgctgccca accagccatggccgaaatag tgatgacgca gtctcc 46 <210> SEQ ID NO 11 <211> LENGTH: 46<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence:Oligo 936 <400>SEQUENCE: 11 tcgctgccca accagccatg gccgatgttg tgatgacaca gtctcc 46 <210>SEQ ID NO 12 <211> LENGTH: 46 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence:Oligo 937 <400> SEQUENCE: 12 tcgctgccca accagccatggccgaaattg tgttgacgca gtctcc 46 <210> SEQ ID NO 13 <211> LENGTH: 46<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence:Oligo 955 <400>SEQUENCE: 13 tcgctgccca accagccatg gccgacatcc agatgatcca gtctcc 46 <210>SEQ ID NO 14 <211> LENGTH: 46 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence:Oligo 956 <400> SEQUENCE: 14 tcgctgccca accagccatggccgatattg tgatgaccca gactcc 46 <210> SEQ ID NO 15 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence:Oligo 973 <400>SEQUENCE: 15 cagcaggcac acaacagagg c 21 <210> SEQ ID NO 16 <211> LENGTH:43 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial Sequence:Oligo 945<400> SEQUENCE: 16 ttacccctgt ggcaaaagcc gaggtgcagc tgttggagtc tgg 43<210> SEQ ID NO 17 <211> LENGTH: 43 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence:Oligo 946 <400> SEQUENCE: 17 ttacccctgtggcaaaagcc gaggtgcagc tggtgcagtc tgg 43 <210> SEQ ID NO 18 <211> LENGTH:43 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial Sequence:Oligo 947<400> SEQUENCE: 18 ttacccctgt ggcaaaagcc caggtgcagc tacagcagtg ggg 43<210> SEQ ID NO 19 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence:Oligo 953 <400> SEQUENCE: 19 gacagatggtgcagccacag t 21 <210> SEQ ID NO 20 <211> LENGTH: 75 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence:Oligo 864 <400>SEQUENCE: 20 atctggcaca tcatatggat aagtttcgtg tacaaaatgc cagacctagaggaattttat 60 ttccagcttg gtccc 75 <210> SEQ ID NO 21 <211> LENGTH: 69<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence:Oligo 862 <400>SEQUENCE: 21 gtgatggtga tggtgatgga tcggagtacc aggttatcga gccctcgatattgaggagac 60 ggtgactga 69 <210> SEQ ID NO 22 <211> LENGTH: 17 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence:Primer 5 <400> SEQUENCE:22 gcaactgttg ggaaggg 17 <210> SEQ ID NO 23 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence:Primer 197 <400>SEQUENCE: 23 tcgctgccca accagccatg 20 <210> SEQ ID NO 24 <211> LENGTH:46 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial Sequence:Primer 869<400> SEQUENCE: 24 gggaccaagc tggaaataaa acgggctgtg gctgcaccat ctgtct 46<210> SEQ ID NO 25 <211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence:Primer 870 <400> SEQUENCE: 25 atctggcacatcatatggat aagactctcc cctgttgaag ctctt 45 <210> SEQ ID NO 26 <211>LENGTH: 38 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence:Primer 867 <400> SEQUENCE: 26 tcagtcaccg tctcctcagc ctccaccaagggcccatc 38 <210> SEQ ID NO 27 <211> LENGTH: 43 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence:Primer 876 <400> SEQUENCE: 27gtgatggtga tggtgatgag atttgggctc tgctttcttg tcc 43 <210> SEQ ID NO 28<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence:Primer 885 <400> SEQUENCE: 28 taagagcggt aagagtgcca g 21 <210>SEQ ID NO 29 <211> LENGTH: 69 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence:Primer 970 <400> SEQUENCE: 29 gtgataaact accgtaaagcttatcgatga taagctgtca attagtgatg gtgatggtga 60 tgagatttg 69 <210> SEQ IDNO 30 <211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence:Decapeptide <400> SEQUENCE: 30 Tyr Pro Tyr Asp ValPro Asp Tyr Ala Ser 1 5 10 <210> SEQ ID NO 31 <211> LENGTH: 47 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence:Table 3 Primer A <400>SEQUENCE: 31 tcgctgccca accagccatg gccagtgcta aagaacttag atctcag 47<210> SEQ ID NO 32 <211> LENGTH: 86 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence:Table 3 Primer B <400> SEQUENCE: 32 gtgataaactaccgcattaa agcttatcga tgataagctg tcaattagtg atggtgatgg 60 tgatgtgaattctcagccct cttcaa 86 <210> SEQ ID NO 33 <211> LENGTH: 21 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence:Table 3 Primer C <400>SEQUENCE: 33 gcaactctct actgtttctc c 21 <210> SEQ ID NO 34 <211> LENGTH:18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial Sequence:Table 3Primer D <400> SEQUENCE: 34 gaggatgacg atgagcgc 18 <210> SEQ ID NO 35<211> LENGTH: 668 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220>FEATURE: <223> OTHER INFORMATION: M1-1L <400> SEQUENCE: 35 aaattgtgttgacgcattcc agccaccctg tctttgtctc caggggaaag agccaccctc 60 tcctgcagggccagtcaggg tgttagcagc tacttagcct ggtaccaaca gaaacctggc 120 caggctcccaggctcctcat ctatgatgca tccaacaggg ccactggcat cccagccagg 180 ttcagtggcagtgggtctgg gacagacttc actctcacca tcagcagcct agagcctgaa 240 gattttgcagtttattactg tcagcagcgt agaactggcc tcggacgttc ggccaaggga 300 ccaaggtggaaatcaaacga actgtggctg caccatctgt cttcatcttc ccgccatctg 360 atgagcagttgaaatctgga actgcctctg ttgtgtgcct gctgaataac ttctatccca 420 gagaggccaaagtacagtgg aaggtggata acgccctcca atcgggtaac tcccaggaga 480 gtgtcacagagcaggacagc aaggacagca cctacagcct cagcagcacc ctgacgctga 540 gcaaagcagactacgagaaa cacaaagtct acgcctgcga agtcacccat cagggcctga 600 gctcgcccgtcacaaagagc ttcaacaggg gagagtctta tccatatgat gtgccagatt 660 atgcgagc 668<210> SEQ ID NO 36 <211> LENGTH: 224 <212> TYPE: PRT <213> ORGANISM:Homo sapiens <220> FEATURE: <223> OTHER INFORMATION: M1-1L <400>SEQUENCE: 36 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser ProGly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Gly Val SerSer Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg LeuLeu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg PheSer Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser LeuGlu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser AsnTrp Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg ThrVal Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu GlnLeu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn PheTyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala LeuGln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp SerLys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser LysAla Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr HisGln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly GluSer Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser 210 215 220 <210> SEQ ID NO37 <211> LENGTH: 678 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(678) <223> OTHERINFORMATION: M1-3L <400> SEQUENCE: 37 gaa ata gtg atg acg cag tct ccagcc acc ctg tct ttg tct cca ggg 48 Glu Ile Val Met Thr Gln Ser Pro AlaThr Leu Ser Leu Ser Pro Gly 1 5 10 15 gaa aga gcc acc ctc tcc tgc agggcc agt cag agt gtt agc agc agc 96 Glu Arg Ala Thr Leu Ser Cys Arg AlaSer Gln Ser Val Ser Ser Ser 20 25 30 tac tta gcc tgg tac cag cag aaa cctggc cag gct ccc agg ctc ctc 144 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro GlyGln Ala Pro Arg Leu Leu 35 40 45 atc tat ggt gca tcc agc agg gcc act ggcatc cca gac agg ttc agt 192 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly IlePro Asp Arg Phe Ser 50 55 60 ggc agt ggg tct ggg aca gac ttc act ctc accatc agc aga ctg gag 240 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr IleSer Arg Leu Glu 65 70 75 80 cct gaa gat ttt gca gtg tat tac tgt cag cagtat ggt agc tca cct 288 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln TyrGly Ser Ser Pro 85 90 95 cca ttc act ttc ggc cct ggg acc aaa gtg gat atcaaa cga act gtg 336 Pro Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile LysArg Thr Val 100 105 110 gct gca cca tct gtc ttc atc ttc ccg cca tct gatgag cag ttg aaa 384 Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp GluGln Leu Lys 115 120 125 tct gga act gcc tct gtt gtg tgc ctg ctg aat aacttc tat ccc aga 432 Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn PheTyr Pro Arg 130 135 140 gag gcc aaa gta cag tgg aag gtg gat aac gcc ctccaa tcg ggt aac 480 Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu GlnSer Gly Asn 145 150 155 160 tcc cag gag agt gtc aca gag cag gac agc aaggac agc acc tac agc 528 Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys AspSer Thr Tyr Ser 165 170 175 ctc agc agc acc ctg acg ctg agc aaa gca gactac gag aaa cac aaa 576 Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp TyrGlu Lys His Lys 180 185 190 gtc tac gcc tgc gaa gtc acc cat cag ggc ctgagc tcg ccc gtc aca 624 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu SerSer Pro Val Thr 195 200 205 aag agc ttc aac agg gga gag tct tat cca tatgat gtg cca gat tat 672 Lys Ser Phe Asn Arg Gly Glu Ser Tyr Pro Tyr AspVal Pro Asp Tyr 210 215 220 gcg agc 678 Ala Ser 225 <210> SEQ ID NO 38<211> LENGTH: 226 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <223>OTHER INFORMATION: M1-3L <400> SEQUENCE: 38 Glu Ile Val Met Thr Gln SerPro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu SerCys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr GlnGln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser SerArg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly ThrAsp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe AlaVal Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95 Pro Phe Thr Phe GlyPro Gly Thr Lys Val Asp Ile Lys Arg Thr Val 100 105 110 Ala Ala Pro SerVal Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 115 120 125 Ser Gly ThrAla Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 130 135 140 Glu AlaLys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 145 150 155 160Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 165 170175 Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 180185 190 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr195 200 205 Lys Ser Phe Asn Arg Gly Glu Ser Tyr Pro Tyr Asp Val Pro AspTyr 210 215 220 Ala Ser 225 <210> SEQ ID NO 39 <211> LENGTH: 672 <212>TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY:CDS <222> LOCATION: (1)..(672) <223> OTHER INFORMATION: M1-4L <400>SEQUENCE: 39 gaa att gtg ttg acg cag tct cca ggc acc ctg tct ttg tct ccaggg 48 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 15 10 15 gaa aga gcc acc ctc tcc tgc agg gcc agt cag agt gtt agc agc agc96 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 2530 tac tta gcc tgg tac cag cag aaa cct ggc cag gct ccc agg ctc cac 144Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu His 35 40 45atc tat ggt gca tcc aga agg gcc act ggc atc cca gac agg ttc agt 192 IleTyr Gly Ala Ser Arg Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 ggcagt ggg tct ggg aca gac ttc act ctc acc atc agc aga ctg gag 240 Gly SerGly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 cctgaa gat ttt gca gtg tat tac tgt cag cag ttt ggt agc tca ttc 288 Pro GluAsp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Gly Ser Ser Phe 85 90 95 act ttcggc cct ggg acc aaa gtg gat atc aaa cga act gtg gct gca 336 Thr Phe GlyPro Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala Ala 100 105 110 cca tctgtc ttc atc ttc ccg cca tct gat gag cag ttg aaa tct gga 384 Pro Ser ValPhe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 act gcctct gtt gtg tgc ctg ctg aat aac ttc tat ccc aga gag gcc 432 Thr Ala SerVal Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 aaa gtacag tgg aag gtg gat aac gcc ctc caa tcg ggt aac tcc cag 480 Lys Val GlnTrp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 gagagt gtc aca gag cag gac agc aag gac agc acc tac agc ctc agc 528 Glu SerVal Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 agcacc ctg acg ctg agc aaa gca gac tac gag aaa cac aaa gtc tac 576 Ser ThrLeu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 gcctgc gaa gtc acc cat cag ggc ctg agc tcg ccc gtc aca aag agc 624 Ala CysGlu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 ttcaac agg gga gag tct tat cca tat gat gtg cca gat tat gcg agc 672 Phe AsnArg Gly Glu Ser Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser 210 215 220<210> SEQ ID NO 40 <211> LENGTH: 224 <212> TYPE: PRT <213> ORGANISM:Homo sapiens <223> OTHER INFORMATION: M1-4L <400> SEQUENCE: 40 Glu IleVal Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 GluArg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 TyrLeu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu His 35 40 45 IleTyr Gly Ala Ser Arg Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 GlySer Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Phe Gly Ser Ser Phe 85 90 95Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala Ala 100 105110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn SerGln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr TyrSer Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu LysHis Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser SerPro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Ser Tyr Pro Tyr AspVal Pro Asp Tyr Ala Ser 210 215 220 <210> SEQ ID NO 41 <211> LENGTH: 678<212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221>NAME/KEY: CDS <222> LOCATION: (1)..(678) <223> OTHER INFORMATION: M1-5L<400> SEQUENCE: 41 gaa ata gtg atg acg cag tct cca ggc acc ctg tct ttgtct cca ggg 48 Glu Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu SerPro Gly 1 5 10 15 gaa aga gcc acc ctc tcc tgc agg gcc agt cag agt gttagc agc agc 96 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val SerSer Ser 20 25 30 tac tta gcc tgg tac cag cag aaa cct ggc cag gct ccc aggctc ctc 144 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg LeuLeu 35 40 45 atc tat ggt gca tcc agc agg gcc act ggc atc cca gac agg ttcagt 192 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser50 55 60 ggc agt ggg tct ggg aca gac ttc act ctc acc atc agc aga ctg gag240 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 6570 75 80 cct gaa gat ttt gca gtg tat tac tgt cag cag tat ggt agc tca cct288 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 8590 95 ata ttc act ttc ggc cct ggg acc aaa gtg gat atc aaa cga act gtg336 Ile Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg Thr Val 100105 110 gct gca cca tct gtc ttc atc ttc ccg cca tct gat gag cag ttg aaa384 Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 115120 125 tct gga act gcc tct gtt gtg tgc ctg ctg aat aac ttc tat ccc aga432 Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 130135 140 gag gcc aaa gta cag tgg aag gtg gat aac gcc ctc caa tcg ggt aac480 Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 145150 155 160 tcc cag gag agt gtc aca gag cag gac agc aag gac agc acc tacagc 528 Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser165 170 175 ctc agc agc acc ctg acg ctg agc aaa gca gac tac gag aaa cacaaa 576 Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys180 185 190 gtc tac gcc tgc gaa gtc acc cat cag ggc ctg agc tcg ccc gtcaca 624 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr195 200 205 aag agc ttc aac agg gga gag tct tat cca tat gat gtg cca gattat 672 Lys Ser Phe Asn Arg Gly Glu Ser Tyr Pro Tyr Asp Val Pro Asp Tyr210 215 220 gcg agc 678 Ala Ser 225 <210> SEQ ID NO 42 <211> LENGTH: 226<212> TYPE: PRT <213> ORGANISM: Homo sapiens <223> OTHER INFORMATION:M1-5L <400> SEQUENCE: 42 Glu Ile Val Met Thr Gln Ser Pro Gly Thr Leu SerLeu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser GlnSer Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly GlnAla Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly IlePro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu ThrIle Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys GlnGln Tyr Gly Ser Ser Pro 85 90 95 Ile Phe Thr Phe Gly Pro Gly Thr Lys ValAsp Ile Lys Arg Thr Val 100 105 110 Ala Ala Pro Ser Val Phe Ile Phe ProPro Ser Asp Glu Gln Leu Lys 115 120 125 Ser Gly Thr Ala Ser Val Val CysLeu Leu Asn Asn Phe Tyr Pro Arg 130 135 140 Glu Ala Lys Val Gln Trp LysVal Asp Asn Ala Leu Gln Ser Gly Asn 145 150 155 160 Ser Gln Glu Ser ValThr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 165 170 175 Leu Ser Ser ThrLeu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 180 185 190 Val Tyr AlaCys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 195 200 205 Lys SerPhe Asn Arg Gly Glu Ser Tyr Pro Tyr Asp Val Pro Asp Tyr 210 215 220 AlaSer 225 <210> SEQ ID NO 43 <211> LENGTH: 672 <212> TYPE: DNA <213>ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS <222>LOCATION: (1)..(672) <223> OTHER INFORMATION: M1-8L <400> SEQUENCE: 43gaa ata gtg atg acg cag tct cca ggc acc ctg tct ttg tct cca ggg 48 GluIle Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15gaa aga gcc acc ctc tcc tgc agg gcc agt cag agt gtt agc agc acc 96 GluArg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Thr 20 25 30 tactta gcc tgg tac cag cag aaa cct ggc cag gct ccc agg ctc ctc 144 Tyr LeuAla Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 atc tatggt gca tcc agc agg gcc act ggc atc cca gac agg ttc agt 192 Ile Tyr GlyAla Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 ggc agt gggtct ggg aca gac ttc act ctc acc atc agc aga ctg gag 240 Gly Ser Gly SerGly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 cct gaa gatttt gca gtg tat tac tgt cag cag tat gtt agc tca ttc 288 Pro Glu Asp PheAla Val Tyr Tyr Cys Gln Gln Tyr Val Ser Ser Phe 85 90 95 act ttc ggc cctggg acc aaa gtg gat atc aaa cga act gtg gct gca 336 Thr Phe Gly Pro GlyThr Lys Val Asp Ile Lys Arg Thr Val Ala Ala 100 105 110 cca tct gtc ttcatc ttc ccg cca tct gat gag cag ttg aaa tct gga 384 Pro Ser Val Phe IlePhe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 act gcc tct gttgtg tgc ctg ctg aat aac ttc tat ccc aga gag gcc 432 Thr Ala Ser Val ValCys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 aaa gta cag tggaag gtg gat aac gcc ctc caa tcg ggt aac tcc cag 480 Lys Val Gln Trp LysVal Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 gag agt gtcaca gag cag gac agc aag gac agc acc tac agc ctc agc 528 Glu Ser Val ThrGlu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 agc acc ctgacg ctg agc aaa gca gac tac gag aaa cac aaa gtc tac 576 Ser Thr Leu ThrLeu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 gcc tgc gaagtc acc cat cag ggc ctg agc tcg ccc gtc aca aag agc 624 Ala Cys Glu ValThr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 ttc aac agggga gag tct tat cca tat gat gtg cca gat tat gcg agc 672 Phe Asn Arg GlyGlu Ser Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser 210 215 220 <210> SEQ IDNO 44 <211> LENGTH: 224 <212> TYPE: PRT <213> ORGANISM: Homo sapiens<223> OTHER INFORMATION: M1-8L <400> SEQUENCE: 44 Glu Ile Val Met ThrGln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala ThrLeu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Thr 20 25 30 Tyr Leu Ala TrpTyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly AlaSer Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly SerGly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu AspPhe Ala Val Tyr Tyr Cys Gln Gln Tyr Val Ser Ser Phe 85 90 95 Thr Phe GlyPro Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro SerVal Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 ThrAla Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys ValTyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val ThrLys Ser 195 200 205 Phe Asn Arg Gly Glu Ser Tyr Pro Tyr Asp Val Pro AspTyr Ala Ser 210 215 220 <210> SEQ ID NO 45 <211> LENGTH: 672 <212> TYPE:DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS<222> LOCATION: (1)..(672) <223> OTHER INFORMATION: M1-10L <400>SEQUENCE: 45 gat gtt gtg atg aca cag tct cca gcc acc ctg tct ttg tct ccaggg 48 Asp Val Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 15 10 15 gaa aga gcc acc ctc tcc tgc agg gcc agt cag agt gtt agc agc tac96 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 2530 tta gcc tgg tac caa cag aaa cct ggc cag gct ccc agg ctc ctc atc 144Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45tat gat gca tcc aac agg gcc act ggc atc cca gcc agg ttc agt ggc 192 TyrAsp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 agtggg tct ggg aca gac ttc act ctc acc atc agc agc cta gag cct 240 Ser GlySer Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 gaagat ttt gca gtt tat tac tgt cag cag cgt agc aac tgg cct ccc 288 Glu AspPhe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro 85 90 95 act ttcggc gga ggg acc aag gtg gag atc aaa cga act gtg gct gca 336 Thr Phe GlyGly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 cca tctgtc ttc atc ttc ccg cca tct gat gag cag ttg aaa tct gga 384 Pro Ser ValPhe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 act gcctct gtt gtg tgc ctg ctg aat aac ttc tat ccc aga gag gcc 432 Thr Ala SerVal Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 aaa gtacag tgg aag gtg gat aac gcc ctc caa tcg ggt aac tcc cag 480 Lys Val GlnTrp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 gagagt gtc aca gag cag gac agc aag gac agc acc tac agc ctc agc 528 Glu SerVal Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 agcacc ctg acg ctg agc aaa gca gac tac gag aaa cac aaa gtc tac 576 Ser ThrLeu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 gcctgc gaa gtc acc cat cag ggc ctg agc tcg ccc gtc aca aag agc 624 Ala CysGlu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 ttcaac agg gga gag tct tat cca tat gat gtg cca gat tat gcg agc 672 Phe AsnArg Gly Glu Ser Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser 210 215 220<210> SEQ ID NO 46 <211> LENGTH: 224 <212> TYPE: PRT <213> ORGANISM:Homo sapiens <223> OTHER INFORMATION: M1-10L <400> SEQUENCE: 46 Asp ValVal Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 GluArg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 LeuAla Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 TyrAsp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 SerGly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn SerGln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr TyrSer Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu LysHis Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser SerPro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Ser Tyr Pro Tyr AspVal Pro Asp Tyr Ala Ser 210 215 220 <210> SEQ ID NO 47 <211> LENGTH: 672<212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221>NAME/KEY: CDS <222> LOCATION: (1)..(672) <223> OTHER INFORMATION: M1-21L<400> SEQUENCE: 47 gcc atc cgg atg acc cag tct cca tcc ttc ctg tct gcatct gta gga 48 Ala Ile Arg Met Thr Gln Ser Pro Ser Phe Leu Ser Ala SerVal Gly 1 5 10 15 gac aga gtc acc atc act tgc cgg gca agt cag agc attagc agc tat 96 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile SerSer Tyr 20 25 30 tta aat tgg tat cag cag aaa cca ggg aaa gcc cct aag ctcctg atc 144 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu LeuIle 35 40 45 tat gct gca tcc agt ttg caa agt ggg gtc cca tca agg ttc agtgtc 192 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Val50 55 60 agt gga tct ggg aca gat ctc act ctc acc atc agc agt ctg caa cct240 Ser Gly Ser Gly Thr Asp Leu Thr Leu Thr Ile Ser Ser Leu Gln Pro 6570 75 80 gaa gat ttt gca act tat tac tgt cag tgt ggt tac agt aca cca ttc288 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Cys Gly Tyr Ser Thr Pro Phe 8590 95 act ttc ggc cct ggg acc aaa gtg gat atc aaa cga act gtg gct gca336 Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala Ala 100105 110 cca tct gtc ttc atc ttc ccg cca tct gat gag cag ttg aaa tct gga384 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115120 125 act gcc tct gtt gtg tgc ctg ctg aat aac ttc tat ccc aga gag gcc432 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130135 140 aaa gta cag tgg aag gtg gat aac gcc ctc caa tcg ggt aac tcc cag480 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145150 155 160 gag agt gtc aca gag cag gac agc aag gac agc acc tac agc ctcagc 528 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser165 170 175 agc acc ctg acg ctg agc aaa gca gac tac gag aaa cac aaa gtctac 576 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr180 185 190 gcc tgc gaa gtc acc cat cag ggc ctg agc tcg ccc gtc aca aagagc 624 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser195 200 205 ttc aac agg gga gag tct tat cca tat gat gtg cca gat tat gcgagc 672 Phe Asn Arg Gly Glu Ser Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser210 215 220 <210> SEQ ID NO 48 <211> LENGTH: 224 <212> TYPE: PRT <213>ORGANISM: Homo sapiens <223> OTHER INFORMATION: M1-21L <400> SEQUENCE:48 Ala Ile Arg Met Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 510 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 2025 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 3540 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Val 5055 60 Ser Gly Ser Gly Thr Asp Leu Thr Leu Thr Ile Ser Ser Leu Gln Pro 6570 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Cys Gly Tyr Ser Thr Pro Phe85 90 95 Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala Ala100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys SerGly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro ArgGlu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser GlyAsn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp SerThr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp TyrGlu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly LeuSer Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Ser Tyr ProTyr Asp Val Pro Asp Tyr Ala Ser 210 215 220 <210> SEQ ID NO 49 <211>LENGTH: 678 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (1)..(678) <223> OTHER INFORMATION:M1-23L <400> SEQUENCE: 49 gaa att gtg ttg acg cag tct cca ggc acc ctgtct ttg tct cca ggg 48 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu SerLeu Ser Pro Gly 1 5 10 15 gaa aga gcc acc ctc tcc tgc agg gcc agt cagagt gtt agc agc agc 96 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln SerVal Ser Ser Ser 20 25 30 tac tta gcc tgg tac cag cag aaa cct ggc cag gctccc agg ctc ctc 144 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala ProArg Leu Leu 35 40 45 atc tat ggt gca tcc agc agg gcc act ggc atc cca gacagg ttc agt 192 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp ArgPhe Ser 50 55 60 ggc agt ggg tct ggg aca gac ttc act ctc acc atc agc agactg gag 240 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg LeuGlu 65 70 75 80 cct gaa gat ttt gca gtg tat tac tgt cag cag tat ggt agctca cct 288 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser SerPro 85 90 95 ccg tac act ttt ggc cag ggg acc aag ctg gag atc aaa cga actgtg 336 Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val100 105 110 gct gca cca tct gtc ttc atc ttc ccg cca tct gat gag cag ttgaaa 384 Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys115 120 125 tct gga act gcc tct gtt gtg tgc ctg ctg aat aac ttc tat cccaga 432 Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg130 135 140 gag gcc aaa gta cag tgg agg gtg gat aac gcc ctc caa tcg ggtaac 480 Glu Ala Lys Val Gln Trp Arg Val Asp Asn Ala Leu Gln Ser Gly Asn145 150 155 160 tcc cag gag agt gtc aca gag cag gac agc aag gac agc acctac agc 528 Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr TyrSer 165 170 175 ctc agc agc acc ctg acg ctg agc aaa gca gac tac gag aaacac aaa 576 Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys HisLys 180 185 190 gtc tac gcc tgc gaa gtc acc cat cag ggc ctg agc tcg cccgtc aca 624 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro ValThr 195 200 205 aag agc ttc aac agg gga gag tct tat cca tat gat gtg ccagat tat 672 Lys Ser Phe Asn Arg Gly Glu Ser Tyr Pro Tyr Asp Val Pro AspTyr 210 215 220 gcg agc 678 Ala Ser 225 <210> SEQ ID NO 50 <211> LENGTH:226 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <223> OTHERINFORMATION: M1-23L <400> SEQUENCE: 50 Glu Ile Val Leu Thr Gln Ser ProGly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser CysArg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln GlnLys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser ArgAla Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr AspPhe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala ValTyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95 Pro Tyr Thr Phe Gly GlnGly Thr Lys Leu Glu Ile Lys Arg Thr Val 100 105 110 Ala Ala Pro Ser ValPhe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 115 120 125 Ser Gly Thr AlaSer Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 130 135 140 Glu Ala LysVal Gln Trp Arg Val Asp Asn Ala Leu Gln Ser Gly Asn 145 150 155 160 SerGln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 165 170 175Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 180 185190 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 195200 205 Lys Ser Phe Asn Arg Gly Glu Ser Tyr Pro Tyr Asp Val Pro Asp Tyr210 215 220 Ala Ser 225 <210> SEQ ID NO 51 <211> LENGTH: 672 <212> TYPE:DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS<222> LOCATION: (1)..(672) <223> OTHER INFORMATION: M1-25L <400>SEQUENCE: 51 gaa att gtg ttg acg cag tct cca ggc acc ctg tct ttg tct ccaggg 48 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 15 10 15 gaa aga gcc acc ctc tcc tgc agg gcc agt cag agt gtt agc agc agc96 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 2530 tac tta gcc tgg tac cag cag aaa cct ggc cag gct ccc agg ctc ctc 144Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45atc tat ggt gca tcc agc agg gcc act ggc atc cca aac agg ttc agt 192 IleTyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asn Arg Phe Ser 50 55 60 ggcagt ggg tct ggg aca gac ttc act ctc acc atc agc aga ctg gag 240 Gly SerGly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 cctgaa gat ttt gca gtg tat tac tgt cag cag tat ggt agc tca ttc 288 Pro GluAsp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Phe 85 90 95 act ttcggc cct ggg acc aaa gtg gat atc aaa cga act gtg gct gca 336 Thr Phe GlyPro Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala Ala 100 105 110 cca tctgtc ttc atc ttc ccg cca tct gat gag cag ttg aaa tct gga 384 Pro Ser ValPhe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 act gcctct gtt gtg tgc ctg ctg aat aac ttc tat ccc aga gag gcc 432 Thr Ala SerVal Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 aaa gtacag tgg aag gtg gat aac gcc ctc caa tcg ggt aac tcc cag 480 Lys Val GlnTrp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 gagagt gtc aca gag cag gac agc aag gac agc acc tac agc ctc agc 528 Glu SerVal Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 agcacc ctg acg ctg agc aaa gca gac tac gag aaa cac aaa gtc tac 576 Ser ThrLeu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 gcctgc gaa gtc acc cat cag ggc ctg agc tcg ccc gtc aca aag agc 624 Ala CysGlu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 ttcaac agg gga gag tct tat cca tat gat gtg cca gat tat gcg agc 672 Phe AsnArg Gly Glu Ser Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser 210 215 220<210> SEQ ID NO 52 <211> LENGTH: 224 <212> TYPE: PRT <213> ORGANISM:Homo sapiens <223> OTHER INFORMATION: M1-25L <400> SEQUENCE: 52 Glu IleVal Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 GluArg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 TyrLeu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 IleTyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asn Arg Phe Ser 50 55 60 GlySer Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Phe 85 90 95Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala Ala 100 105110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn SerGln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr TyrSer Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu LysHis Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser SerPro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Ser Tyr Pro Tyr AspVal Pro Asp Tyr Ala Ser 210 215 220 <210> SEQ ID NO 53 <211> LENGTH: 675<212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221>NAME/KEY: CDS <222> LOCATION: (1)..(675) <223> OTHER INFORMATION: M1-1H<400> SEQUENCE: 53 cag gtg cag ctg gtg gag tct ggg gga ggc gtg gtc cagcct ggg aag 48 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln ProGly Lys 1 5 10 15 tcc ctg aga ctc tcc tgt gca gcg tct gaa ttc acc atcagt tac tat 96 Ser Leu Arg Leu Ser Cys Ala Ala Ser Glu Phe Thr Ile SerTyr Tyr 20 25 30 ggc atg cac tgg gtc cgc cag gtt cca ggc aag ggg ctg gagtgg gtg 144 Gly Met His Trp Val Arg Gln Val Pro Gly Lys Gly Leu Glu TrpVal 35 40 45 gca gct gtc tgg tat gat gaa agt act aca tat tct cca gac tccgtg 192 Ala Ala Val Trp Tyr Asp Glu Ser Thr Thr Tyr Ser Pro Asp Ser Val50 55 60 aag ggc cga ttc acc atc tcc aga gac gat tcc aag aac acg ctg tat240 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr 6570 75 80 ctg caa atg aac agc ctg aga gcc gag gac acg gct gtg tat tac tgt288 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 8590 95 gcg aga gat agg gtg ggc ctc ttt gac tac tgg ggc cag gga acc ctg336 Ala Arg Asp Arg Val Gly Leu Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100105 110 gtc acc gtc tcc tca gcc tcc acc aag ggc cca tcg gtc ttc ccc ctg384 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115120 125 gca ccc tcc tcc aag agc acc tct ggg ggc aca gcg gcc ctg ggc tgc432 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130135 140 ctg gtc aag gac tac ttc ccc gaa ccg gtg acg gtg tcg tgg aac tca480 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145150 155 160 ggc gcc ctg acc agc ggc gtg cac acc ttc ccg gct gtc cta cagtcc 528 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser165 170 175 tca gga ctc tac tcc ctc agc agc gtg gtg acc gtg ccc tcc agcagc 576 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser180 185 190 ttg ggc acc cag acc tac atc tgc aac gtg aat cac aag ccc agcaac 624 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn195 200 205 acc aag gtg gac aag aaa gca gag ccc aaa tct cat cac cat caccat 672 Thr Lys Val Asp Lys Lys Ala Glu Pro Lys Ser His His His His His210 215 220 cac 675 His 225 <210> SEQ ID NO 54 <211> LENGTH: 225 <212>TYPE: PRT <213> ORGANISM: Homo sapiens <223> OTHER INFORMATION: M1-1H<400> SEQUENCE: 54 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val GlnPro Gly Lys 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Glu Phe ThrIle Ser Tyr Tyr 20 25 30 Gly Met His Trp Val Arg Gln Val Pro Gly Lys GlyLeu Glu Trp Val 35 40 45 Ala Ala Val Trp Tyr Asp Glu Ser Thr Thr Tyr SerPro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser LysAsn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp ThrAla Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Val Gly Leu Phe Asp Tyr TrpGly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys GlyPro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser GlyGly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro GluPro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser GlyVal His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr SerLeu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr GlnThr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys ValAsp Lys Lys Ala Glu Pro Lys Ser His His His His His 210 215 220 His 225<210> SEQ ID NO 55 <211> LENGTH: 677 <212> TYPE: DNA <213> ORGANISM:Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION:(3)..(677) <223> OTHER INFORMATION: M1-3H <400> SEQUENCE: 55 cc gat gtgcag ctg gtg cag tct ggg gga ggc gtg gtc cag cct ggg 47 Asp Val Gln LeuVal Gln Ser Gly Gly Gly Val Val Gln Pro Gly 1 5 10 15 agg tcc ctg agactc tcc tgt gca gcg tct gga ttc acc ttc agt tac 95 Arg Ser Leu Arg LeuSer Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr 20 25 30 tat ggc atg cac tgggtc cgc cag gct cca ggc aag ggg ctg gag tgg 143 Tyr Gly Met His Trp ValArg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 gtg aca ctt ata acc tatgat gga gat aat aaa tac tat gca gac tcc 191 Val Thr Leu Ile Thr Tyr AspGly Asp Asn Lys Tyr Tyr Ala Asp Ser 50 55 60 gtg aag ggc cga ttc acc atctcc aga gac aat tcc aag aac acg ctg 239 Val Lys Gly Arg Phe Thr Ile SerArg Asp Asn Ser Lys Asn Thr Leu 65 70 75 tat ctg caa atg aac agc ctg agagcc gag gac acg gct gtg tat tac 287 Tyr Leu Gln Met Asn Ser Leu Arg AlaGlu Asp Thr Ala Val Tyr Tyr 80 85 90 95 tgt gcg aga gac ggg atc ggg tacttt gac tat tgg ggc cag gga acc 335 Cys Ala Arg Asp Gly Ile Gly Tyr PheAsp Tyr Trp Gly Gln Gly Thr 100 105 110 ctg gtc acc gtc tcc tca gcc tccacc aag ggc cca tcg gtc ttc ccc 383 Leu Val Thr Val Ser Ser Ala Ser ThrLys Gly Pro Ser Val Phe Pro 115 120 125 ctg gca ccc tcc tcc aag agc acctct ggg ggc aca gcg gcc ctg ggc 431 Leu Ala Pro Ser Ser Lys Ser Thr SerGly Gly Thr Ala Ala Leu Gly 130 135 140 tgc ctg gtc aag gac tac ttc cccgaa ccg gtg acg gtg tcg tgg aac 479 Cys Leu Val Lys Asp Tyr Phe Pro GluPro Val Thr Val Ser Trp Asn 145 150 155 tca ggc gcc ctg acc agc ggc gtgcac acc ttc ccg gct gtc cta cag 527 Ser Gly Ala Leu Thr Ser Gly Val HisThr Phe Pro Ala Val Leu Gln 160 165 170 175 tcc tca gga ctc tac tcc ctcagc agc gtg gtg acc gtg ccc tcc agc 575 Ser Ser Gly Leu Tyr Ser Leu SerSer Val Val Thr Val Pro Ser Ser 180 185 190 agc ttg ggc acc cag acc tacatc tgc aac gtg aat cac aag ccc agc 623 Ser Leu Gly Thr Gln Thr Tyr IleCys Asn Val Asn His Lys Pro Ser 195 200 205 aac acc aag gtg gac aag aaagca gag ccc aaa tct cat cac cat cac 671 Asn Thr Lys Val Asp Lys Lys AlaGlu Pro Lys Ser His His His His 210 215 220 cat cac 677 His His 225<210> SEQ ID NO 56 <211> LENGTH: 225 <212> TYPE: PRT <213> ORGANISM:Homo sapiens <223> OTHER INFORMATION: M1-3H <400> SEQUENCE: 56 Asp ValGln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 SerLeu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Tyr 20 25 30 GlyMet His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 ThrLeu Ile Thr Tyr Asp Gly Asp Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 LysGly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Gly Ile Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp AsnSer 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala ValLeu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr ValPro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val AsnHis Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Lys Ala Glu Pro LysSer His His His His His 210 215 220 His 225 <210> SEQ ID NO 57 <211>LENGTH: 675 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (1)..(675) <223> OTHER INFORMATION:M1-4H <400> SEQUENCE: 57 cag gtg cag ctg gtg gag tct ggg gga ggc gtg gtccag cct ggg aag 48 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val GlnPro Gly Lys 1 5 10 15 tcc ctg aga ctc tcc tgt gca gcg tct gga ttc accttc agt tac tat 96 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr PheSer Tyr Tyr 20 25 30 ggc atg cac tgg gtc cgc cag gtt cca ggc aag ggg ctggag tgg gtg 144 Gly Met His Trp Val Arg Gln Val Pro Gly Lys Gly Leu GluTrp Val 35 40 45 gca gct gtc tgg tat gat gga agt act aca tat tct cca gactcc gtg 192 Ala Ala Val Trp Tyr Asp Gly Ser Thr Thr Tyr Ser Pro Asp SerVal 50 55 60 aag ggc cga ttc acc atc tcc aga gac gat tcc aag aac acg ctgtat 240 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr65 70 75 80 ctg caa atg aac agc ctg aga gcc gag gac acg gct gtg tat tactgt 288 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys85 90 95 gcg aga gat agg gtg ggc ctc ttt gac tac tgg ggc cag gga acc ctg336 Ala Arg Asp Arg Val Gly Leu Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100105 110 gtc acc gtc tcc tca gcc tcc acc aag ggc cca tcg gtc ttc ccc ctg384 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115120 125 gca ccc tcc tcc aag agc acc tct ggg ggc aca gcg gcc ctg ggc tgc432 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130135 140 ctg gtc aag gac tac ttc ccc gaa ccg gtg acg gtg tcg tgg aac tca480 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145150 155 160 ggc gcc ctg acc agc ggc gtg cac acc ttc ccg gct gtc cta cagtcc 528 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser165 170 175 tca gga ctc tac tcc ctc agc agc gtg gtg acc gtg ccc tcc agcagc 576 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser180 185 190 ttg ggc acc cag acc tac atc tgc aac gtg aat cac aag ccc agcaac 624 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn195 200 205 acc aag gtg gac aag aaa gca ggg ccc aaa tct cat cac cat caccat 672 Thr Lys Val Asp Lys Lys Ala Gly Pro Lys Ser His His His His His210 215 220 cac 675 His 225 <210> SEQ ID NO 58 <211> LENGTH: 225 <212>TYPE: PRT <213> ORGANISM: Homo sapiens <223> OTHER INFORMATION: M1-4H<400> SEQUENCE: 58 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val GlnPro Gly Lys 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe ThrPhe Ser Tyr Tyr 20 25 30 Gly Met His Trp Val Arg Gln Val Pro Gly Lys GlyLeu Glu Trp Val 35 40 45 Ala Ala Val Trp Tyr Asp Gly Ser Thr Thr Tyr SerPro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser LysAsn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp ThrAla Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Val Gly Leu Phe Asp Tyr TrpGly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys GlyPro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser GlyGly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro GluPro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser GlyVal His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr SerLeu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr GlnThr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys ValAsp Lys Lys Ala Gly Pro Lys Ser His His His His His 210 215 220 His 225<210> SEQ ID NO 59 <211> LENGTH: 675 <212> TYPE: DNA <213> ORGANISM:Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION:(1)..(675) <223> OTHER INFORMATION: M1-5H <400> SEQUENCE: 59 cag gtg cagctg gtg gag tct ggg gga ggc gtg gtc cag cct ggg agg 48 Gln Val Gln LeuVal Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 tcc ctg agactc tcc tgt gca gcg tct gga ttt acc ttc agt tac tat 96 Ser Leu Arg LeuSer Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Tyr 20 25 30 ggc atg cac tgggtc cgc cag gct cca ggc aag ggg ctg gag tgg gtg 144 Gly Met His Trp ValArg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 aca ctt ata acc tatgat gga gat aat aaa tac tat gca gac tcc gtg 192 Thr Leu Ile Thr Tyr AspGly Asp Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 aag ggc cga ttc acc atctcc aga gac aat tcc aag aac acg ctg tat 240 Lys Gly Arg Phe Thr Ile SerArg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 ctg caa atg aac agc ctgaga gcc gag gac acg gct gtg tat tac tgt 288 Leu Gln Met Asn Ser Leu ArgAla Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gcg aga gac ggg atc ggg tacttt gac tat tgg ggc cag gga acc ctg 336 Ala Arg Asp Gly Ile Gly Tyr PheAsp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 gtc acc gtc tcc tca gcc tccacc aag ggc cca tcg gtc ttc ccc ctg 384 Val Thr Val Ser Ser Ala Ser ThrLys Gly Pro Ser Val Phe Pro Leu 115 120 125 gca ccc tcc tcc aag agc acctct ggg ggc aca gcg gcc ctg ggc tgc 432 Ala Pro Ser Ser Lys Ser Thr SerGly Gly Thr Ala Ala Leu Gly Cys 130 135 140 ctg gtc aag gac tac ttc cccgaa ccg gtg acg gtg tcg tgg aac tca 480 Leu Val Lys Asp Tyr Phe Pro GluPro Val Thr Val Ser Trp Asn Ser 145 150 155 160 ggc gcc ctg acc agc ggcgtg cac acc ttc ccg gct gtc cta cag tcc 528 Gly Ala Leu Thr Ser Gly ValHis Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 tca gga ctc tac tcc ctcagc agc gtg gtg acc gtg ccc tcc agc agc 576 Ser Gly Leu Tyr Ser Leu SerSer Val Val Thr Val Pro Ser Ser Ser 180 185 190 ttg ggc acc cag acc tacatc tgc aac gtg aat cac aag ccc agc aac 624 Leu Gly Thr Gln Thr Tyr IleCys Asn Val Asn His Lys Pro Ser Asn 195 200 205 acc aag gtg gac aag aaagca gag ccc aaa tct cat cac cat cac cat 672 Thr Lys Val Asp Lys Lys AlaGlu Pro Lys Ser His His His His His 210 215 220 cac 675 His 225 <210>SEQ ID NO 60 <211> LENGTH: 225 <212> TYPE: PRT <213> ORGANISM: Homosapiens <223> OTHER INFORMATION: M1-5H <400> SEQUENCE: 60 Gln Val GlnLeu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser LeuArg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Tyr 20 25 30 Gly MetHis Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Thr LeuIle Thr Tyr Asp Gly Asp Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys GlyArg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 LeuGln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 AlaArg Asp Gly Ile Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val LeuGln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val ProSer Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn HisLys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Lys Ala Glu Pro Lys SerHis His His His His 210 215 220 His 225 <210> SEQ ID NO 61 <211> LENGTH:675 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221>NAME/KEY: CDS <222> LOCATION: (1)..(675) <223> OTHER INFORMATION: M1-8H<400> SEQUENCE: 61 cag gtg cag ctg gtg cag tct ggg gga ggc gtg gtc cagcct ggg aag 48 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln ProGly Lys 1 5 10 15 tcc ctg aaa ctc tcc tgt gca gcg tct gga ttc acc ttcagt tac tat 96 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe SerTyr Tyr 20 25 30 ggc atg cac tgg gtc cgc cag gct cca ggc aag ggg ctg gagtgg gtg 144 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu TrpVal 35 40 45 gca gct gta tgg tat gat gga agt aac aca tac tct cca gac tccgtg 192 Ala Ala Val Trp Tyr Asp Gly Ser Asn Thr Tyr Ser Pro Asp Ser Val50 55 60 aag ggc cga ttc acc atc tcc aga gac gat tcc aag aac acg gtg tat240 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Val Tyr 6570 75 80 ctg caa atg aac agc ctg aga gcc gag gac acg gct gtg tat tac tgt288 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 8590 95 gcg aga gat agg gtg ggc ctc ttt gac tac tgg ggc cag gga acc ctg336 Ala Arg Asp Arg Val Gly Leu Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100105 110 gtc acc gtc tcc tca gcc tcc acc aag ggc cca tcg gtc ttc ccc ctg384 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115120 125 gca ccc tcc tcc aag agc acc tct ggg ggc aca gcg gcc ctg ggc tgc432 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130135 140 ctg gtc aag gac tac ttc ccc gaa ccg gtg acg gtg tcg tgg aac tca480 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145150 155 160 ggc gcc ctg acc agc ggc gtg cac acc ttc ccg gct gtc cta cagtcc 528 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser165 170 175 tca gga ctc tac tcc ctc agc agc gtg gtg acc gtg ccc tcc agcagc 576 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser180 185 190 ttg ggc acc cag acc tac atc tgc aac gtg aat cac aag ccc agcaac 624 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn195 200 205 acc aag gtg gac aag aaa gca gag ccc aaa tct cat cac cat caccat 672 Thr Lys Val Asp Lys Lys Ala Glu Pro Lys Ser His His His His His210 215 220 cac 675 His 225 <210> SEQ ID NO 62 <211> LENGTH: 225 <212>TYPE: PRT <213> ORGANISM: Homo sapiens <223> OTHER INFORMATION: M1-8H<400> SEQUENCE: 62 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val GlnPro Gly Lys 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe ThrPhe Ser Tyr Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys GlyLeu Glu Trp Val 35 40 45 Ala Ala Val Trp Tyr Asp Gly Ser Asn Thr Tyr SerPro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser LysAsn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp ThrAla Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Val Gly Leu Phe Asp Tyr TrpGly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys GlyPro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser GlyGly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro GluPro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser GlyVal His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr SerLeu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr GlnThr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys ValAsp Lys Lys Ala Glu Pro Lys Ser His His His His His 210 215 220 His 225<210> SEQ ID NO 63 <211> LENGTH: 708 <212> TYPE: DNA <213> ORGANISM:Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION:(1)..(708) <223> OTHER INFORMATION: M1-10H <400> SEQUENCE: 63 cag gtgcag ctg gtg cag tct ggg gga ggc ttg gta cat cct ggg ggg 48 Gln Val GlnLeu Val Gln Ser Gly Gly Gly Leu Val His Pro Gly Gly 1 5 10 15 tcc ctgaga ctc tcc tgt gaa ggc tct gga ttc atc ttc agg aac cat 96 Ser Leu ArgLeu Ser Cys Glu Gly Ser Gly Phe Ile Phe Arg Asn His 20 25 30 cct ata cactgg gtt cgc cag gct cca gga aaa ggt ctg gag tgg gta 144 Pro Ile His TrpVal Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 tca gtt agt ggtatt ggt ggt gac aca tac tat gca gac tcc gtg aag 192 Ser Val Ser Gly IleGly Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 ggc cga ttc tcc atctcc aga gac aat gcc aag aac tcc ttg tat ctt 240 Gly Arg Phe Ser Ile SerArg Asp Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 caa atg aac agc ctgaga gcc gag gac atg gct gtg tat tac tgt gca 288 Gln Met Asn Ser Leu ArgAla Glu Asp Met Ala Val Tyr Tyr Cys Ala 85 90 95 aga gaa tat tac tat ggttcg ggg agt tat cgc gtt gac tac tac tac 336 Arg Glu Tyr Tyr Tyr Gly SerGly Ser Tyr Arg Val Asp Tyr Tyr Tyr 100 105 110 tac ggt atg gac gtc tggggc caa ggg acc acg gtc acc gtc tcc tca 384 Tyr Gly Met Asp Val Trp GlyGln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 gcc tcc acc aag ggc ccatcg gtc ttc ccc ctg gca ccc tcc tcc aag 432 Ala Ser Thr Lys Gly Pro SerVal Phe Pro Leu Ala Pro Ser Ser Lys 130 135 140 agc acc tct ggg ggc acagcg gcc ctg ggc tgc ctg gtc aag gac tac 480 Ser Thr Ser Gly Gly Thr AlaAla Leu Gly Cys Leu Val Lys Asp Tyr 145 150 155 160 ttc ccc gaa ccg gtgacg gtg tcg tgg aac tca ggc gcc ctg acc agc 528 Phe Pro Glu Pro Val ThrVal Ser Trp Asn Ser Gly Ala Leu Thr Ser 165 170 175 ggc gtg cac acc ttcccg gct gtc cta cag tcc tca gga ctc tac tcc 576 Gly Val His Thr Phe ProAla Val Leu Gln Ser Ser Gly Leu Tyr Ser 180 185 190 ctc agc agc gtg gtgacc gtg ccc tcc agc agc ttg ggc acc cag acc 624 Leu Ser Ser Val Val ThrVal Pro Ser Ser Ser Leu Gly Thr Gln Thr 195 200 205 tac atc tgc aac gtgaat cac aag ccc agc aac acc aag gtg gac aag 672 Tyr Ile Cys Asn Val AsnHis Lys Pro Ser Asn Thr Lys Val Asp Lys 210 215 220 aaa gca gag ccc aaatct cat cac cat cac cat cac 708 Lys Ala Glu Pro Lys Ser His His His HisHis His 225 230 235 <210> SEQ ID NO 64 <211> LENGTH: 236 <212> TYPE: PRT<213> ORGANISM: Homo sapiens <223> OTHER INFORMATION: M1-10H <400>SEQUENCE: 64 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val His Pro GlyGly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Gly Ser Gly Phe Ile Phe ArgAsn His 20 25 30 Pro Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu GluTrp Val 35 40 45 Ser Val Ser Gly Ile Gly Gly Asp Thr Tyr Tyr Ala Asp SerVal Lys 50 55 60 Gly Arg Phe Ser Ile Ser Arg Asp Asn Ala Lys Asn Ser LeuTyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala Val TyrTyr Cys Ala 85 90 95 Arg Glu Tyr Tyr Tyr Gly Ser Gly Ser Tyr Arg Val AspTyr Tyr Tyr 100 105 110 Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr ValThr Val Ser Ser 115 120 125 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro LeuAla Pro Ser Ser Lys 130 135 140 Ser Thr Ser Gly Gly Thr Ala Ala Leu GlyCys Leu Val Lys Asp Tyr 145 150 155 160 Phe Pro Glu Pro Val Thr Val SerTrp Asn Ser Gly Ala Leu Thr Ser 165 170 175 Gly Val His Thr Phe Pro AlaVal Leu Gln Ser Ser Gly Leu Tyr Ser 180 185 190 Leu Ser Ser Val Val ThrVal Pro Ser Ser Ser Leu Gly Thr Gln Thr 195 200 205 Tyr Ile Cys Asn ValAsn His Lys Pro Ser Asn Thr Lys Val Asp Lys 210 215 220 Lys Ala Glu ProLys Ser His His His His His His 225 230 235 <210> SEQ ID NO 65 <211>LENGTH: 675 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (1)..(675) <223> OTHER INFORMATION:M1-21H <400> SEQUENCE: 65 cag gtg cag ctg gtg cag tct ggg gga ggc gtggtc cag cct ggg aag 48 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val ValGln Pro Gly Lys 1 5 10 15 tcc ctg aga ctc tcc tgt gca gcg tct gga ttcacc ttc agt tac tat 96 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe ThrPhe Ser Tyr Tyr 20 25 30 ggc atg cac tgg gtc cgc cag gtt cca ggc aag gggctg gag tgg gtg 144 Gly Met His Trp Val Arg Gln Val Pro Gly Lys Gly LeuGlu Trp Val 35 40 45 gca gct gtc tgg tat gat gga agt act aca tat tct ccagac tcc gtg 192 Ala Ala Val Trp Tyr Asp Gly Ser Thr Thr Tyr Ser Pro AspSer Val 50 55 60 aag ggc cga ttc acc atc tcc aga gac gat tcc aag aac acgctg tat 240 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr LeuTyr 65 70 75 80 ctg caa atg agc agc ctg aga gcc gag gac acg gct gtg tattac tgt 288 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr TyrCys 85 90 95 gcg aga gat agg gtg ggc ctc ttt gac tac tgg ggc cag gga accctg 336 Ala Arg Asp Arg Val Gly Leu Phe Asp Tyr Trp Gly Gln Gly Thr Leu100 105 110 gtc acc gtc tcc tca gcc tcc acc aag ggc cca tcg gtc ttc cccctg 384 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu115 120 125 gca ccc tcc tcc aag agc acc tct ggg ggc aca gcg gcc ctg ggctgc 432 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys130 135 140 ctg gtc aag gac tac ttc ccc gaa ccg gtg acg gtg tcg tgg aactca 480 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser145 150 155 160 ggc gcc ctg acc agc ggc gtg cac acc ttc ccg gct gtc ctacag tcc 528 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu GlnSer 165 170 175 tca gga ctc tac tcc ctc agc agc gtg gtg acc gtg ccc tccagc agc 576 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser SerSer 180 185 190 ttg ggc acc cag acc tac atc tgc aac gtg aat cac aag cccagc aac 624 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro SerAsn 195 200 205 acc aag gtg gac aag aaa gca gag ccc aaa tct cat cac catcac cat 672 Thr Lys Val Asp Lys Lys Ala Glu Pro Lys Ser His His His HisHis 210 215 220 cac 675 His 225 <210> SEQ ID NO 66 <211> LENGTH: 225<212> TYPE: PRT <213> ORGANISM: Homo sapiens <223> OTHER INFORMATION:M1-21H <400> SEQUENCE: 66 Gln Val Gln Leu Val Gln Ser Gly Gly Gly ValVal Gln Pro Gly Lys 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser GlyPhe Thr Phe Ser Tyr Tyr 20 25 30 Gly Met His Trp Val Arg Gln Val Pro GlyLys Gly Leu Glu Trp Val 35 40 45 Ala Ala Val Trp Tyr Asp Gly Ser Thr ThrTyr Ser Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp AspSer Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala GluAsp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Val Gly Leu Phe AspTyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser ThrLys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser ThrSer Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr PhePro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu ThrSer Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly LeuTyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu GlyThr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 ThrLys Val Asp Lys Lys Ala Glu Pro Lys Ser His His His His His 210 215 220His 225 <210> SEQ ID NO 67 <211> LENGTH: 675 <212> TYPE: DNA <213>ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS <222>LOCATION: (1)..(675) <223> OTHER INFORMATION: M1-23H <400> SEQUENCE: 67cag gtg cag ctg gtg cag tct ggg gga ggc gtg gtc cag cct ggg agg 48 GlnVal Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15tcc ctg aga ctc tcc tgt gca gcg tct gga ttc acc ttc agt aac tat 96 SerLeu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 ggcatg cac tgg gtc cgc cag gct cca ggc aag ggg ctg gag tgg gtg 144 Gly MetHis Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 gca gctata tgg tat gat gga agt aaa aca tac aat gca gac tcc gtg 192 Ala Ala IleTrp Tyr Asp Gly Ser Lys Thr Tyr Asn Ala Asp Ser Val 50 55 60 aag ggc cgattc acc atc tcc aga gac aat tcc aag aac acg ctg tat 240 Lys Gly Arg PheThr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 ctg caa atgaac agc ctg aga gcc gag gac acg gct gtg tat tac tgt 288 Leu Gln Met AsnSer Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gcg aga gat gggata ggc tac ttt gac tac tgg ggc cag gga acc ctg 336 Ala Arg Asp Gly IleGly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 gtc acc gtc tcctca gcc tcc acc aag ggc cca tcg gtc ttc ccc ctg 384 Val Thr Val Ser SerAla Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 gca ccc tcc tccaag agc acc tct ggg ggc aca gcg gcc ctg ggc tgc 432 Ala Pro Ser Ser LysSer Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 ctg gtc aag gactac ttc ccc gaa ccg gtg acg gtg tcg tgg aac tca 480 Leu Val Lys Asp TyrPhe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 ggc gcc ctgacc agc ggc gtg cac acc ttc ccg gct gtc cta cag tcc 528 Gly Ala Leu ThrSer Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 tca gga ctctac tcc ctc agc agc gtg gtg acc gtg ccc tcc agc agc 576 Ser Gly Leu TyrSer Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 ttg ggc acccag acc tac atc tgc aac gtg aat cac aag ccc agc aac 624 Leu Gly Thr GlnThr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 acc aag gtggac aag aaa gca gag ccc aaa tct cat cac cat cac cat 672 Thr Lys Val AspLys Lys Ala Glu Pro Lys Ser His His His His His 210 215 220 cac 675 His225 <210> SEQ ID NO 68 <211> LENGTH: 225 <212> TYPE: PRT <213> ORGANISM:Homo sapiens <223> OTHER INFORMATION: M1-23H <400> SEQUENCE: 68 Gln ValGln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 SerLeu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 GlyMet His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 AlaAla Ile Trp Tyr Asp Gly Ser Lys Thr Tyr Asn Ala Asp Ser Val 50 55 60 LysGly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Gly Ile Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp AsnSer 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala ValLeu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr ValPro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val AsnHis Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Lys Ala Glu Pro LysSer His His His His His 210 215 220 His 225 <210> SEQ ID NO 69 <211>LENGTH: 675 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (1)..(675) <223> OTHER INFORMATION:M1-25H <400> SEQUENCE: 69 cag gtg cag ctg gtg gag tct ggg gga ggc ttggtc cag cct ggg ggg 48 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu ValGln Pro Gly Gly 1 5 10 15 tcc ctg aga ctc tcc tgt gca gcg tct gga ttcacc ttc agt tac tat 96 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe ThrPhe Ser Tyr Tyr 20 25 30 ggc atg cac tgg gtc cgc cag gtt cca ggc aag gggctg gag tgg gtg 144 Gly Met His Trp Val Arg Gln Val Pro Gly Lys Gly LeuGlu Trp Val 35 40 45 gca gct gtc tgg tat gat gga agt act aca tat cct ccagac tcc gtg 192 Ala Ala Val Trp Tyr Asp Gly Ser Thr Thr Tyr Pro Pro AspSer Val 50 55 60 aag ggc cga ttc acc atc tcc aga gac gat tcc aag aac acgctg tat 240 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr LeuTyr 65 70 75 80 ctg caa atg aac agc ctg aga gcc gag gac acg gct gtt tattac tgt 288 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr TyrCys 85 90 95 gcg aga gat agg gtg ggc ctc ttt gac tac tgg ggc cag gga accctg 336 Ala Arg Asp Arg Val Gly Leu Phe Asp Tyr Trp Gly Gln Gly Thr Leu100 105 110 gtc acc gtc tcc tca gcc tcc acc aag ggc cca tcg gtc ttc cccctg 384 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu115 120 125 gca ccc tcc tcc aag agc acc tct ggg ggc aca gcg gcc ctg ggctgc 432 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys130 135 140 ctg gtc aag gac tac ttc ccc gaa ccg gtg acg gtg tcg tgg aactca 480 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser145 150 155 160 ggc gcc ctg acc agc ggc gtg cac acc ttc ccg gct gtc ctacag tcc 528 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu GlnSer 165 170 175 tca gga ctc tac tcc ctc agc agc gtg gtg acc gtg ccc tccagc agc 576 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser SerSer 180 185 190 ttg ggc acc cag acc tac atc tgc aac gtg aat cac aag cccagc aac 624 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro SerAsn 195 200 205 acc aag gtg gac aag aaa gca gag ccc aaa tct cat cac catcac cat 672 Thr Lys Val Asp Lys Lys Ala Glu Pro Lys Ser His His His HisHis 210 215 220 cac 675 His 225 <210> SEQ ID NO 70 <211> LENGTH: 225<212> TYPE: PRT <213> ORGANISM: Homo sapiens <223> OTHER INFORMATION:M1-25H <400> SEQUENCE: 70 Gln Val Gln Leu Val Glu Ser Gly Gly Gly LeuVal Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser GlyPhe Thr Phe Ser Tyr Tyr 20 25 30 Gly Met His Trp Val Arg Gln Val Pro GlyLys Gly Leu Glu Trp Val 35 40 45 Ala Ala Val Trp Tyr Asp Gly Ser Thr ThrTyr Pro Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp AspSer Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala GluAsp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Val Gly Leu Phe AspTyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser ThrLys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser ThrSer Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr PhePro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu ThrSer Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly LeuTyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu GlyThr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 ThrLys Val Asp Lys Lys Ala Glu Pro Lys Ser His His His His His 210 215 220His 225 <210> SEQ ID NO 71 <211> LENGTH: 678 <212> TYPE: DNA <213>ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS <222>LOCATION: (1)..(678) <223> OTHER INFORMATION: M2-11L <400> SEQUENCE: 71gaa ata gtg atg acg cag tct cca ggc acc ctg tct ttg tct cca ggg 48 GluIle Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15gaa aga gcc acc ctc tcc tgc agg gcc agt cag ggt gtt agc agc agc 96 GluArg Ala Thr Leu Ser Cys Arg Ala Ser Gln Gly Val Ser Ser Ser 20 25 30 tactta gcc tgg tac cag cag aaa cct ggc cag gct ccc agg ctc ctc 144 Tyr LeuAla Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 atc tatggt gca tcc agc agg gcc act ggc atc cca gac agg ttc agt 192 Ile Tyr GlyAla Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 ggc agt gggtct ggg aca gac ttc act ctc acc atc agc aga ctg gag 240 Gly Ser Gly SerGly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 cct gaa gatttt gca gtg tat tac tgt cag cag tat ggt agc tca cct 288 Pro Glu Asp PheAla Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95 cca ttc act ttcggc cct ggg acc aaa gtg gat atc aaa cga act gtg 336 Pro Phe Thr Phe GlyPro Gly Thr Lys Val Asp Ile Lys Arg Thr Val 100 105 110 gct gca cca tctgtc ttc atc ttc ccg cca tct gat gag cag ttg aga 384 Ala Ala Pro Ser ValPhe Ile Phe Pro Pro Ser Asp Glu Gln Leu Arg 115 120 125 tct gga act gcctct gtt gtg tgc ctg ctg aat aac ttc tat ccc aga 432 Ser Gly Thr Ala SerVal Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 130 135 140 gag gcc aaa gtacag tgg aag gtg gat aac gcc ctc caa tcg ggt aac 480 Glu Ala Lys Val GlnTrp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 145 150 155 160 tcc cag gagagt gtc aca gag cag gac agc aag gac agc acc tac agc 528 Ser Gln Glu SerVal Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 165 170 175 ctc agc agcacc ctg acg ctg agc aaa gca gac tac gag aaa cac aaa 576 Leu Ser Ser ThrLeu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 180 185 190 gtc tac gcctgc gaa gtc acc cat cag ggc ctg agc tcg ccc gtc aca 624 Val Tyr Ala CysGlu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 195 200 205 aag agc ttcaac agg gga gag tct tat cca tat gat gtg cca gat tat 672 Lys Ser Phe AsnArg Gly Glu Ser Tyr Pro Tyr Asp Val Pro Asp Tyr 210 215 220 gcg agc 678Ala Ser 225 <210> SEQ ID NO 72 <211> LENGTH: 226 <212> TYPE: PRT <213>ORGANISM: Homo sapiens <223> OTHER INFORMATION: M2-11L <400> SEQUENCE:72 Glu Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 510 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Gly Val Ser Ser Ser 2025 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 3540 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 5055 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 6570 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro85 90 95 Pro Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg Thr Val100 105 110 Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln LeuArg 115 120 125 Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe TyrPro Arg 130 135 140 Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu GlnSer Gly Asn 145 150 155 160 Ser Gln Glu Ser Val Thr Glu Gln Asp Ser LysAsp Ser Thr Tyr Ser 165 170 175 Leu Ser Ser Thr Leu Thr Leu Ser Lys AlaAsp Tyr Glu Lys His Lys 180 185 190 Val Tyr Ala Cys Glu Val Thr His GlnGly Leu Ser Ser Pro Val Thr 195 200 205 Lys Ser Phe Asn Arg Gly Glu SerTyr Pro Tyr Asp Val Pro Asp Tyr 210 215 220 Ala Ser 225 <210> SEQ ID NO73 <211> LENGTH: 678 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(678) <223> OTHERINFORMATION: M2-12L <400> SEQUENCE: 73 gaa ata gtg atg acg cag tct ccaggc acc ctg tct ttg tct cca ggg 48 Glu Ile Val Met Thr Gln Ser Pro GlyThr Leu Ser Leu Ser Pro Gly 1 5 10 15 gaa aga gcc acc ctc tcc tgc agggcc agt cag ggt gtt agc agc agc 96 Glu Arg Ala Thr Leu Ser Cys Arg AlaSer Gln Gly Val Ser Ser Ser 20 25 30 tac tta gcc tgg tac cag cag aaa cctggc cag gct ccc agg ctc ctc 144 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro GlyGln Ala Pro Arg Leu Leu 35 40 45 atc tat ggt gca tcc agc agg gcc act ggcatc cca gac agg ttc agt 192 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly IlePro Asp Arg Phe Ser 50 55 60 ggc agt ggg tct ggg aca gac ttc act ctc accatc agc agc cta gag 240 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr IleSer Ser Leu Glu 65 70 75 80 cct gaa gat ttt gca gtg tat tac tgt cag cagtat ggt agc tca cct 288 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln TyrGly Ser Ser Pro 85 90 95 ccg tac act ttt ggc cag ggg acc aag ctg gag atcaaa cga act gtg 336 Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile LysArg Thr Val 100 105 110 gct gca cca tct gtc ttc atc ttc ccg cca tct gatgag cag ttg aaa 384 Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp GluGln Leu Lys 115 120 125 tct gga act gcc tct gtt gtg tgc ctg ctg aat aacttc tat ccc aga 432 Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn PheTyr Pro Arg 130 135 140 gag gcc aaa gta cag tgg aag gtg gat aac gcc ctccaa tcg ggt aac 480 Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu GlnSer Gly Asn 145 150 155 160 tcc cag gag agt gtc aca gag cag gac agc aaggac agc acc tac agc 528 Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys AspSer Thr Tyr Ser 165 170 175 ctc agc agc acc ctg acg ctg agc aaa gca gactac gag aaa cac aaa 576 Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp TyrGlu Lys His Lys 180 185 190 gtc tac gcc tgc gaa gtc acc cat cag ggc ctgagc tcg ccc gtc aca 624 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu SerSer Pro Val Thr 195 200 205 aag agc ttc aac agg gga gag tct tat cca tatgat gtg cca gat tat 672 Lys Ser Phe Asn Arg Gly Glu Ser Tyr Pro Tyr AspVal Pro Asp Tyr 210 215 220 gcg agc 678 Ala Ser 225 <210> SEQ ID NO 74<211> LENGTH: 226 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <223>OTHER INFORMATION: M2-12L <400> SEQUENCE: 74 Glu Ile Val Met Thr Gln SerPro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu SerCys Arg Ala Ser Gln Gly Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr GlnGln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser SerArg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly ThrAsp Phe Thr Leu Thr Ile Ser Ser Leu Glu 65 70 75 80 Pro Glu Asp Phe AlaVal Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95 Pro Tyr Thr Phe GlyGln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val 100 105 110 Ala Ala Pro SerVal Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 115 120 125 Ser Gly ThrAla Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 130 135 140 Glu AlaLys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 145 150 155 160Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 165 170175 Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 180185 190 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr195 200 205 Lys Ser Phe Asn Arg Gly Glu Ser Tyr Pro Tyr Asp Val Pro AspTyr 210 215 220 Ala Ser 225 <210> SEQ ID NO 75 <211> LENGTH: 672 <212>TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY:CDS <222> LOCATION: (1)..(672) <223> OTHER INFORMATION: M2-16L <400>SEQUENCE: 75 gaa ata gtg atg acg cag tct cca ggc acc ctg tct ttg tct ccaggg 48 Glu Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 15 10 15 gaa aga gcc acc ctc tcc tgc agg gcc agt cag agt gtt agc agc agc96 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 2530 tac tta gcc tgg tac cag cag aaa cct ggc cag gct ccc agg ctc ctc 144Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45atc tat ggt gca tcc agc agg gcc act ggc atc cca gac agg ttc agt 192 IleTyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 gtcagt ggg tct ggg aca gac ttc act ctc acc atc agc aga ctg gag 240 Val SerGly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 cctgaa gat ttt gca gtg tat tac tgt cag cag tat ggt agc tca ttc 288 Pro GluAsp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Phe 85 90 95 act ttcggc cct ggg acc aaa gtg gat atc aaa cga act gtg gct gca 336 Thr Phe GlyPro Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala Ala 100 105 110 cca tctgtc ttc atc ttc ccg cca tct gat gag cag ttg aaa tct gga 384 Pro Ser ValPhe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 act gcctct gtt gtg tgc ctg ctg aat aac ttc tat ccc aga gag gcc 432 Thr Ala SerVal Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 aaa gtacag tgg aag gtg gat aac gcc ctc caa tcg ggt aac tcc cag 480 Lys Val GlnTrp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 gagagt gtc aca gag cag gac agc aag gac agc acc tac agc ctc agc 528 Glu SerVal Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 agcacc ctg acg ctg agc aaa gca gac tac gag aaa cac aaa gtc tac 576 Ser ThrLeu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 gcctgc gaa gtc acc cat cag ggc ctg agc tcg ccc gtc aca aag agc 624 Ala CysGlu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 ttcaac agg gga gag tct tat cca tat gat gtg cca gat tat gcg agc 672 Phe AsnArg Gly Glu Ser Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser 210 215 220<210> SEQ ID NO 76 <211> LENGTH: 224 <212> TYPE: PRT <213> ORGANISM:Homo sapiens <223> OTHER INFORMATION: M2-16L <400> SEQUENCE: 76 Glu IleVal Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 GluArg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 TyrLeu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 IleTyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 ValSer Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Phe 85 90 95Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala Ala 100 105110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn SerGln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr TyrSer Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu LysHis Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser SerPro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Ser Tyr Pro Tyr AspVal Pro Asp Tyr Ala Ser 210 215 220 <210> SEQ ID NO 77 <211> LENGTH: 672<212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221>NAME/KEY: CDS <222> LOCATION: (1)..(672) <223> OTHER INFORMATION: M2-18L<400> SEQUENCE: 77 gaa ata gtg atg acg cag tct cca ggc acc ctg tct ttgtct cca ggg 48 Glu Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu SerPro Gly 1 5 10 15 gaa aga gcc acc ctc tcc tgc agg gcc agt cag agt gttagc agc acc 96 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val SerSer Thr 20 25 30 tac tta gcc tgg tac cag cag aaa cct ggc cag gct ccc aggctc ctc 144 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg LeuLeu 35 40 45 atc tat ggt gca tcc agc agg gcc act ggc atc cca gac agg ttcagt 192 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser50 55 60 ggc agt ggg tct ggg aca gac ttc act ctc acc atc agc aga ctg gag240 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 6570 75 80 cct gaa gat ttt gca gtg tat tac tgt cag cag tat gtt agc tca ttc288 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Val Ser Ser Phe 8590 95 act ttc ggc cct ggg acc aaa gtg gat atc aaa cga act gtg gct gca336 Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala Ala 100105 110 cca tct gtc ttc atc ttc ccg cca tct gat gag cag ttg aaa tct gga384 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115120 125 act gcc tct gtt gtg tgc ctg ctg aat aac ttc tat ccc aga gag gcc432 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130135 140 aaa gta cag tgg aag gtg gat aac gcc ctc caa tcg ggt aac tcc cag480 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145150 155 160 gag agt gtc aca gag cag gac agc aag gac agc acc tac agc ctcagc 528 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser165 170 175 agc acc ctg acg ctg agc aaa gca gac tac gag aaa cac aaa gtctac 576 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr180 185 190 gcc tgc gaa gtc acc cat cag ggc ctg agc tcg ccc gtc aca aagagc 624 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser195 200 205 ttc aac agg gga gag tct tat cca tat gat gtg cca gat tat gcgagc 672 Phe Asn Arg Gly Glu Ser Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser210 215 220 <210> SEQ ID NO 78 <211> LENGTH: 224 <212> TYPE: PRT <213>ORGANISM: Homo sapiens <223> OTHER INFORMATION: M2-18L <400> SEQUENCE:78 Glu Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 510 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Thr 2025 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 3540 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 5055 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 6570 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Val Ser Ser Phe85 90 95 Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala Ala100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys SerGly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro ArgGlu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser GlyAsn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp SerThr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp TyrGlu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly LeuSer Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Ser Tyr ProTyr Asp Val Pro Asp Tyr Ala Ser 210 215 220 <210> SEQ ID NO 79 <211>LENGTH: 678 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (1)..(678) <223> OTHER INFORMATION:M2-20L <400> SEQUENCE: 79 gaa ata gtg atg acg cag tct cca ggc acc ctgtct ttg tct cca ggg 48 Glu Ile Val Met Thr Gln Ser Pro Gly Thr Leu SerLeu Ser Pro Gly 1 5 10 15 gaa aga gcc acc ctc tcc tgc agg gcc agt cagagt gtt agc agc agc 96 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln SerVal Ser Ser Ser 20 25 30 tac tta gcc tgg tac cag cag aaa cct ggc cag gctccc agg ctc ctc 144 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala ProArg Leu Leu 35 40 45 atc tac ggt gca tcc agg agg gcc act ggc atc cca gacagg ttc agt 192 Ile Tyr Gly Ala Ser Arg Arg Ala Thr Gly Ile Pro Asp ArgPhe Ser 50 55 60 ggc agt ggg tct ggg aca gac ttc act ctc acc atc agc agactg gag 240 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg LeuGlu 65 70 75 80 cct gaa gat ttt gca gtg tat tac tgt cag cag tat ggt agctca ccc 288 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser SerPro 85 90 95 atg tac act ttt ggc cag ggg acc aag ctg gag atc aaa cga actgtg 336 Met Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val100 105 110 gct gca cca tct gtc ttc atc ttc ccg cca tct gat gag cag ttgaaa 384 Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys115 120 125 tct gga act gcc tct gtt gtg tgc ctg ctg aat aac ttc tat cccaga 432 Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg130 135 140 gag gcc aaa gta cag tgg aag gtg gat aac gcc ctc caa tcg ggtaac 480 Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn145 150 155 160 tcc cag gag agt gtc aca gag cag gac agc aag gac agc acctac agc 528 Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr TyrSer 165 170 175 ctc agc agc acc ctg acg ctg agc aaa gca gac tac gag aaacac aaa 576 Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys HisLys 180 185 190 gtc tac gcc tgc gaa gtc acc cat cag ggc ctg agc tcg cccgtc aca 624 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro ValThr 195 200 205 aag agc ttc aac agg gga gag tct tat cca tat gat gtg ccagat tat 672 Lys Ser Phe Asn Arg Gly Glu Ser Tyr Pro Tyr Asp Val Pro AspTyr 210 215 220 gcg agc 678 Ala Ser 225 <210> SEQ ID NO 80 <211> LENGTH:226 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <223> OTHERINFORMATION: M2-20L <400> SEQUENCE: 80 Glu Ile Val Met Thr Gln Ser ProGly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser CysArg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln GlnLys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Arg ArgAla Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr AspPhe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala ValTyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95 Met Tyr Thr Phe Gly GlnGly Thr Lys Leu Glu Ile Lys Arg Thr Val 100 105 110 Ala Ala Pro Ser ValPhe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 115 120 125 Ser Gly Thr AlaSer Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 130 135 140 Glu Ala LysVal Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 145 150 155 160 SerGln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 165 170 175Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 180 185190 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 195200 205 Lys Ser Phe Asn Arg Gly Glu Ser Tyr Pro Tyr Asp Val Pro Asp Tyr210 215 220 Ala Ser 225 <210> SEQ ID NO 81 <211> LENGTH: 672 <212> TYPE:DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS<222> LOCATION: (1)..(672) <223> OTHER INFORMATION: M2-31L <400>SEQUENCE: 81 gaa att gtg ttg acg cag tct cca gcc acc ctg tct ttg tct ccaggg 48 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 15 10 15 gaa aga gcc acc ctc tcc tgc agg gcc agt cag agt gtt agc agc tac96 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 2530 tta gcc tgg tac caa cag aaa cct ggc cag gct ccc agg ctc ctc atc 144Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45tat gat gca tcc aac agg gcc act ggc atc cca gcc agg ttc agt ggc 192 TyrAsp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 agtggg tct ggg aca gac ttc act ctc acc atc agc agc cta gag cct 240 Ser GlySer Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 gaagat ttt gca gtt tat tac tgt cag cag cgt acg aac tgg cct cgg 288 Glu AspPhe Ala Val Tyr Tyr Cys Gln Gln Arg Thr Asn Trp Pro Arg 85 90 95 acg ttcggc caa ggg acc aag gtg gaa atc aaa cga act gtg gct gca 336 Thr Phe GlyGln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 cca tctgtc ttc atc ttc ccg cca tct gat gag cag ttg aaa tct gga 384 Pro Ser ValPhe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 act gcctct gtt gtg tgc ctg ctg aat aac ttc tat ccc aga gag gcc 432 Thr Ala SerVal Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 aaa gtacag tgg aag gtg gat aac gcc ctc caa tcg ggt aac tcc cag 480 Lys Val GlnTrp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 gagagt gtc aca gag cag gac agc aag gac agc acc tac agc ctc agc 528 Glu SerVal Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 agcacc ctg acg ctg agc aaa gca gac tac gag aaa cac aaa gtc tac 576 Ser ThrLeu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 gcctgc gaa gtc acc cat cag ggc ctg agc tcg ccc gtc aca aag agc 624 Ala CysGlu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 ttcaac agg gga gag tct tat cca tat gat gtg cca gat tat gcg agc 672 Phe AsnArg Gly Glu Ser Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser 210 215 220<210> SEQ ID NO 82 <211> LENGTH: 224 <212> TYPE: PRT <213> ORGANISM:Homo sapiens <223> OTHER INFORMATION: M2-31L <400> SEQUENCE: 82 Glu IleVal Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 GluArg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 LeuAla Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 TyrAsp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 SerGly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Thr Asn Trp Pro Arg 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn SerGln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr TyrSer Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu LysHis Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser SerPro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Ser Tyr Pro Tyr AspVal Pro Asp Tyr Ala Ser 210 215 220 <210> SEQ ID NO 83 <211> LENGTH: 672<212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221>NAME/KEY: CDS <222> LOCATION: (1)..(672) <223> OTHER INFORMATION: M2-32L<400> SEQUENCE: 83 gaa att gtg ttg acg cag tct cca gcc acc ctg tct ttgtct cca ggg 48 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu SerPro Gly 1 5 10 15 gaa aga gcc acc ctc tcc tgc agg gcc agt cag agt gttagc agc tac 96 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val SerSer Tyr 20 25 30 tta gcc tgg tac caa cag aaa cct ggc cag gct ccc agg ctcctc atc 144 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu LeuIle 35 40 45 tat gat gca tcc aac agg gcc gct ggc atc cca gcc agg ttc agtggc 192 Tyr Asp Ala Ser Asn Arg Ala Ala Gly Ile Pro Ala Arg Phe Ser Gly50 55 60 agt ggg tct ggg aca gac ttc act ctc acc atc agc agc cta gag cct240 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 6570 75 80 gaa gat ttt gca gtt tat tac tgt cag caa cgt aac aac tgg cct ctc288 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Asn Asn Trp Pro Leu 8590 95 act ttc ggc gga ggg acc aag gtg gag atc aaa cga act gtg gct gca336 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100105 110 cca tct gtc ttc atc ttc ccg cca tct gat gag cag ttg aaa tct gga384 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115120 125 act gcc tct gtt gtg tgc ctg ctg aat aac ttc tat ccc aga gag gcc432 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130135 140 aaa gta cag tgg aag gtg gat aac gcc ctc caa tcg ggt aac tcc cag480 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145150 155 160 gag agt gtc aca gag cag gac agc aag gac agc acc tac agc ctcagc 528 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser165 170 175 agc acc ctg acg ctg agc aaa gca gac tac gag aaa cac aaa gtctac 576 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr180 185 190 gcc tgc gaa gtc acc cat cag ggc ctg agc tcg ccc gtc aca aagagc 624 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser195 200 205 ttc aac agg gga gag tct tat cca tat gat gtg cca gat tat gcgagc 672 Phe Asn Arg Gly Glu Ser Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser210 215 220 <210> SEQ ID NO 84 <211> LENGTH: 224 <212> TYPE: PRT <213>ORGANISM: Homo sapiens <223> OTHER INFORMATION: M2-32L <400> SEQUENCE:84 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 510 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 2025 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 3540 45 Tyr Asp Ala Ser Asn Arg Ala Ala Gly Ile Pro Ala Arg Phe Ser Gly 5055 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 6570 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Asn Asn Trp Pro Leu85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys SerGly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro ArgGlu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser GlyAsn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp SerThr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp TyrGlu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly LeuSer Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Ser Tyr ProTyr Asp Val Pro Asp Tyr Ala Ser 210 215 220 <210> SEQ ID NO 85 <211>LENGTH: 678 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (1)..(678) <223> OTHER INFORMATION:M2-33L <400> SEQUENCE: 85 gaa att gtg ttg acg cag tct cca ggc acc ctgtct ttg tct cca ggg 48 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu SerLeu Ser Pro Gly 1 5 10 15 gaa aga gcc acc ctc tcc tgc agg gcc agt cagagt gtt agc agc agc 96 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln SerVal Ser Ser Ser 20 25 30 tac tta gcc tgg tac cag cag aaa cct ggc cag gctccc agg ctc ctc 144 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala ProArg Leu Leu 35 40 45 atc tat ggt gca tcc agc agg gcc act ggc atc cca gacagg ttc agt 192 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp ArgPhe Ser 50 55 60 ggc agt ggg tct ggg aca gac ttc act ctc acc atc agc agactg gag 240 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg LeuGlu 65 70 75 80 cct gaa gat ttt gca gtg tat tac tgt cag cag tat ggt agctca cct 288 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser SerPro 85 90 95 ccg tac act ttt ggc cag ggg acc aag ctg gag atc aaa cga actgtg 336 Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val100 105 110 gct gca cca tct gtc ttc atc ttc ccg cca tct gat gag cag ttgaaa 384 Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys115 120 125 tct gga act gcc tct gtt gtg tgc ctg ctg aat aac ttc tat cccaga 432 Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg130 135 140 gag gcc aaa gta cag tgg aag gtg gat aac gcc ctc caa tcg ggtaac 480 Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn145 150 155 160 tcc cag gag agt gtc aca gag cag gac agc aag gac agc acctac agc 528 Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr TyrSer 165 170 175 ctc agc agc acc ctg acg ctg agc aaa gca gac tac gag aaacac aaa 576 Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys HisLys 180 185 190 gtc tac gcc tgc gaa gtc acc cat cag ggc ctg agc tcg cccgtc aca 624 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro ValThr 195 200 205 aag agc ttc aac agg gga gag tct tat cca tat gat gtg ccagat tat 672 Lys Ser Phe Asn Arg Gly Glu Ser Tyr Pro Tyr Asp Val Pro AspTyr 210 215 220 gcg agc 678 Ala Ser 225 <210> SEQ ID NO 86 <211> LENGTH:226 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <223> OTHERINFORMATION: M2-33L <400> SEQUENCE: 86 Glu Ile Val Leu Thr Gln Ser ProGly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser CysArg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln GlnLys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser ArgAla Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr AspPhe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala ValTyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95 Pro Tyr Thr Phe Gly GlnGly Thr Lys Leu Glu Ile Lys Arg Thr Val 100 105 110 Ala Ala Pro Ser ValPhe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 115 120 125 Ser Gly Thr AlaSer Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 130 135 140 Glu Ala LysVal Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 145 150 155 160 SerGln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 165 170 175Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 180 185190 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 195200 205 Lys Ser Phe Asn Arg Gly Glu Ser Tyr Pro Tyr Asp Val Pro Asp Tyr210 215 220 Ala Ser 225 <210> SEQ ID NO 87 <211> LENGTH: 672 <212> TYPE:DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS<222> LOCATION: (1)..(672) <223> OTHER INFORMATION: M2-34L <400>SEQUENCE: 87 gaa att gtg ttg acg cag tct cca gcc acc ctg tct ttg tct ccaggg 48 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 15 10 15 gaa aga gcc acc ctc tcc tgc agg gcc agt cag agt gtt agc agc tac96 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 2530 tta gcc tgg tac caa cag aaa cct ggc cag gct ccc agg ctc ctc atc 144Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45tat gat gca tcc aac agg gcc act ggc atc cca gcc agg ttc agt ggc 192 TyrAsp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 agtggg tct ggg aca gac ttc act ctc acc atc agc agc cta gag cct 240 Ser GlySer Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 gaagat ttt gca gtt tat tac tgt cag cag cgt acg aac tgg cct cgg 288 Glu AspPhe Ala Val Tyr Tyr Cys Gln Gln Arg Thr Asn Trp Pro Arg 85 90 95 acg ttcggc caa ggg acc aag gtg gaa atc aaa cga act gtg gct gca 336 Thr Phe GlyGln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 cca tctgtc ttc atc ttc ccg cca tct gat gag cag ttg aaa tct gga 384 Pro Ser ValPhe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 act gcctct gtt gtg tgc ctg ctg aat aac ttc tat ccc aga gag gcc 432 Thr Ala SerVal Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 aaa gtacag tgg aag gtg gat aac gcc ctc caa tcg ggt aac tcc cag 480 Lys Val GlnTrp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 gagagt gtc aca gag cag gac agc aag gac agc acc tac agc ctc agc 528 Glu SerVal Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 agcacc ctg acg ctg agc aaa gca gac tac gag aaa cac aaa gtc tac 576 Ser ThrLeu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 gcctgc gaa gtc acc cat cag ggc ctg agc tcg ccc gtc aca aag agc 624 Ala CysGlu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 ttcaac agg gga gag tct tat cca tat gat gtg cca gat tat gcg agc 672 Phe AsnArg Gly Glu Ser Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser 210 215 220<210> SEQ ID NO 88 <211> LENGTH: 224 <212> TYPE: PRT <213> ORGANISM:Homo sapiens <223> OTHER INFORMATION: M2-34L <400> SEQUENCE: 88 Glu IleVal Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 GluArg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 LeuAla Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 TyrAsp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 SerGly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Thr Asn Trp Pro Arg 85 90 95Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn SerGln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr TyrSer Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu LysHis Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser SerPro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Ser Tyr Pro Tyr AspVal Pro Asp Tyr Ala Ser 210 215 220 <210> SEQ ID NO 89 <211> LENGTH: 672<212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221>NAME/KEY: CDS <222> LOCATION: (1)..(672) <223> OTHER INFORMATION: M2-35L<400> SEQUENCE: 89 gaa att gtg ttg acg cag tct cca gcc acc ctg tct ttgtct cca ggg 48 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu SerPro Gly 1 5 10 15 gaa aga gcc acc ctc tcc tgc agg gcc agt cag agt gttagc agc tac 96 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val SerSer Tyr 20 25 30 tta gcc tgg tac caa cag aaa cct ggc cag gct ccc agg ctcctc atc 144 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu LeuIle 35 40 45 tat gat gca tcc aac agg gcc act ggc atc cca gcc agg ttc agtggc 192 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly50 55 60 agt ggg tct ggg aca gac ttc act ctc acc atc agc agc cta gag cct240 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 6570 75 80 gaa gat ttt gca gtt tat tac tgt cag cag cgt acg aac tgg cct cgg288 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Thr Asn Trp Pro Arg 8590 95 acg ttc ggc caa ggg acc aag gtg gaa atc aaa cga act gtg gct gca336 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100105 110 cca tct gtc ttc atc ttc ccg cca tct gat gag cag ttg aaa tct gga384 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115120 125 act gcc tct gtt gtg tgc ctg ctg aat aac ttc tat ccc aga gag gcc432 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130135 140 aaa gta cag tgg aag gtg gat aac gcc ctc caa tcg ggt aac tcc cag480 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145150 155 160 gag agt gtc aca gag cag gac agc aag gac agc acc tac agc ctcagc 528 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser165 170 175 agc acc ctg acg ctg agc aaa gca gac tac gag aaa cac aaa gtctac 576 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr180 185 190 gcc tgc gaa gtc acc cat cag ggc ctg agc tcg ccc gtc aca aagagc 624 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser195 200 205 ttc aac agg gga gag tct tat cca tat gat gtg cca gat tat gcgagc 672 Phe Asn Arg Gly Glu Ser Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser210 215 220 <210> SEQ ID NO 90 <211> LENGTH: 224 <212> TYPE: PRT <213>ORGANISM: Homo sapiens <223> OTHER INFORMATION: M2-35L <400> SEQUENCE:90 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 510 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 2025 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 3540 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 5055 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 6570 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Thr Asn Trp Pro Arg85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys SerGly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro ArgGlu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser GlyAsn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp SerThr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp TyrGlu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly LeuSer Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Ser Tyr ProTyr Asp Val Pro Asp Tyr Ala Ser 210 215 220 <210> SEQ ID NO 91 <211>LENGTH: 675 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (1)..(675) <223> OTHER INFORMATION:M2-11H <400> SEQUENCE: 91 cag gtg cag ctg gtg gag tct ggg gga ggc gtggtc cag cct ggg agg 48 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val ValGln Pro Gly Arg 1 5 10 15 tcc ctg aga ctc tcc tgt gca gcg tct gga tttacc ttc agt tac tat 96 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe ThrPhe Ser Tyr Tyr 20 25 30 ggc atg cac tgg gtc cgc cag gct cca ggc aag gggctg gag tgg gtg 144 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly LeuGlu Trp Val 35 40 45 aca ctt ata acc tat gat gga gat aat aaa tac tat gcagac tcc gtg 192 Thr Leu Ile Thr Tyr Asp Gly Asp Asn Lys Tyr Tyr Ala AspSer Val 50 55 60 aag ggc cga ttc acc atc tcc aga gac aat tcc aag aac acgctg tat 240 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr LeuTyr 65 70 75 80 ctg caa atg aac agc ctg aga gcc gag gac acg gct gtg tattac tgt 288 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr TyrCys 85 90 95 gcg aga gac ggg atc ggg tac ttt gac tat tgg ggc cag gga accctg 336 Ala Arg Asp Gly Ile Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu100 105 110 gtc acc gtc tcc tca gcc tcc acc aag ggc cca tcg gtc ttc cccctg 384 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu115 120 125 gca ccc tcc tcc aag agc acc tct ggg ggc aca gcg gcc ctg ggctgc 432 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys130 135 140 ctg gtc aag gac tac ttc ccc gaa ccg gtg acg gtg tcg tgg aactca 480 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser145 150 155 160 ggc gcc ctg acc agc ggc gtg cac acc ttc ccg gct gtc ctacag tcc 528 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu GlnSer 165 170 175 tca gga ctc tac tcc ctc agc agc gtg gtg acc gtg ccc tccagc agc 576 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser SerSer 180 185 190 ttg ggc acc cag acc tac atc tgc aac gtg aat cac aag cccagc aac 624 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro SerAsn 195 200 205 acc aag gtg gac aag aaa gca gag ccc aaa tct cat cac catcac cat 672 Thr Lys Val Asp Lys Lys Ala Glu Pro Lys Ser His His His HisHis 210 215 220 cac 675 His 225 <210> SEQ ID NO 92 <211> LENGTH: 225<212> TYPE: PRT <213> ORGANISM: Homo sapiens <223> OTHER INFORMATION:M2-11H <400> SEQUENCE: 92 Gln Val Gln Leu Val Glu Ser Gly Gly Gly ValVal Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser GlyPhe Thr Phe Ser Tyr Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro GlyLys Gly Leu Glu Trp Val 35 40 45 Thr Leu Ile Thr Tyr Asp Gly Asp Asn LysTyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp AsnSer Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala GluAsp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Ile Gly Tyr Phe AspTyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser ThrLys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser ThrSer Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr PhePro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu ThrSer Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly LeuTyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu GlyThr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 ThrLys Val Asp Lys Lys Ala Glu Pro Lys Ser His His His His His 210 215 220His 225 <210> SEQ ID NO 93 <211> LENGTH: 675 <212> TYPE: DNA <213>ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS <222>LOCATION: (1)..(675) <223> OTHER INFORMATION: M2-12H <400> SEQUENCE: 93gat gtg cag ctg gtg gag tct ggg gga ggc gtg gtc cat cct ggg agg 48 AspVal Gln Leu Val Glu Ser Gly Gly Gly Val Val His Pro Gly Arg 1 5 10 15tcc ctg aga ctc tcc tgt gca gcg tct gga ttt acc ttc agt tac tat 96 SerLeu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Tyr 20 25 30 ggcatg cac tgg gtc cgc cag gct cca ggc aag ggg ctg gaa tgg atg 144 Gly MetHis Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 aca cttata tcc tat gat gga gat aat aaa tac tat gca gac tcc gtg 192 Thr Leu IleSer Tyr Asp Gly Asp Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 aag ggc cgattc acc atc tcc aga gaa aat tcc aag aac acg ctg tat 240 Lys Gly Arg PheThr Ile Ser Arg Glu Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 ctg caa atgaac agt ctg aga gcc gag gac acg gct gtg tat tac tgt 288 Leu Gln Met AsnSer Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gcg aga gac gggatc ggg tac ttt gac tat tgg ggc cag gga acc ctg 336 Ala Arg Asp Gly IleGly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 gtc acc gtc tcctca gcc tcc acc aag ggc cca tcg gtc ttc ccc ctg 384 Val Thr Val Ser SerAla Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 gca ccc tcc tccaag agc acc tct ggg ggc aca gcg gcc ctg ggc tgc 432 Ala Pro Ser Ser LysSer Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 ctg gtc aag gactac ttc ccc gaa ccg gtg acg gtg tcg tgg aac tca 480 Leu Val Lys Asp TyrPhe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 ggc gcc ctgacc agc ggc gtg cac acc ttc ccg gct gtc cta cag tcc 528 Gly Ala Leu ThrSer Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 tca gga ctctac tcc ctc agc agc gtg gtg acc gtg ccc tcc agc agc 576 Ser Gly Leu TyrSer Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 ttg ggc acccag acc tac atc tgc aac gtg aat cac aag ccc agc agc 624 Leu Gly Thr GlnThr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Ser 195 200 205 acc aag gtggac aag aaa gca gag ccc aaa tct cat cac cat cac cat 672 Thr Lys Val AspLys Lys Ala Glu Pro Lys Ser His His His His His 210 215 220 cac 675 His225 <210> SEQ ID NO 94 <211> LENGTH: 225 <212> TYPE: PRT <213> ORGANISM:Homo sapiens <223> OTHER INFORMATION: M2-12H <400> SEQUENCE: 94 Asp ValGln Leu Val Glu Ser Gly Gly Gly Val Val His Pro Gly Arg 1 5 10 15 SerLeu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Tyr 20 25 30 GlyMet His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 ThrLeu Ile Ser Tyr Asp Gly Asp Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 LysGly Arg Phe Thr Ile Ser Arg Glu Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Gly Ile Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp AsnSer 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala ValLeu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr ValPro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val AsnHis Lys Pro Ser Ser 195 200 205 Thr Lys Val Asp Lys Lys Ala Glu Pro LysSer His His His His His 210 215 220 His 225 <210> SEQ ID NO 95 <211>LENGTH: 675 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (1)..(675) <223> OTHER INFORMATION:M2-16H <400> SEQUENCE: 95 cag gtg cag ctg gtg cag tct ggg gga ggc gtggtc cag cct ggg aag 48 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val ValGln Pro Gly Lys 1 5 10 15 tcc ctg aga ctc tcc tgt gca gcg tct gga ttcagc ttg agt tac tat 96 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe SerLeu Ser Tyr Tyr 20 25 30 ggc atg cac tgg gtc cgc cag gtt cca ggc aag gggctg gag tgg gtg 144 Gly Met His Trp Val Arg Gln Val Pro Gly Lys Gly LeuGlu Trp Val 35 40 45 gca gct gtc tgg tat gat gga agt act aga tat tct ccagac tcc gtg 192 Ala Ala Val Trp Tyr Asp Gly Ser Thr Arg Tyr Ser Pro AspSer Val 50 55 60 aag ggc cga ttc acc atc tcc aga gac gat tcc aag aac acgctg tat 240 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr LeuTyr 65 70 75 80 ctg caa atg aac agc ctg aga gcc gag gac acg gct gtg tattac tgt 288 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr TyrCys 85 90 95 gcg aga gat agg gtg ggc ctc ttt gac tac tgg ggc cag gga accctg 336 Ala Arg Asp Arg Val Gly Leu Phe Asp Tyr Trp Gly Gln Gly Thr Leu100 105 110 gtc acc gtc tcc tca gcc tcc acc aag ggc cca tcg gtc ttc cccctg 384 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu115 120 125 gca ccc tcc tcc aag agc acc tct ggg ggc aca gcg gcc ctg ggctgc 432 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys130 135 140 ctg gtc aag gac tac ttc ccc gaa ccg gtg acg gtg tcg tgg aactca 480 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser145 150 155 160 ggc gcc ctg acc agc ggc gtg cac acc ttc ccg gct gtc ctacag tcc 528 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu GlnSer 165 170 175 tca gga ctc tac tcc ctc agc agc gtg gtg acc gtg ccc tccagc agc 576 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser SerSer 180 185 190 ttg ggc acc cag acc tac atc tgc aac gtg aat cac aag cccagc aac 624 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro SerAsn 195 200 205 acc aag gtg gac aag aaa gca gag ccc aaa tct cat cac catcac cat 672 Thr Lys Val Asp Lys Lys Ala Glu Pro Lys Ser His His His HisHis 210 215 220 cac 675 His 225 <210> SEQ ID NO 96 <211> LENGTH: 225<212> TYPE: PRT <213> ORGANISM: Homo sapiens <223> OTHER INFORMATION:M2-16H <400> SEQUENCE: 96 Gln Val Gln Leu Val Gln Ser Gly Gly Gly ValVal Gln Pro Gly Lys 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser GlyPhe Ser Leu Ser Tyr Tyr 20 25 30 Gly Met His Trp Val Arg Gln Val Pro GlyLys Gly Leu Glu Trp Val 35 40 45 Ala Ala Val Trp Tyr Asp Gly Ser Thr ArgTyr Ser Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp AspSer Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala GluAsp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Val Gly Leu Phe AspTyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser ThrLys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser ThrSer Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr PhePro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu ThrSer Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly LeuTyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu GlyThr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 ThrLys Val Asp Lys Lys Ala Glu Pro Lys Ser His His His His His 210 215 220His 225 <210> SEQ ID NO 97 <211> LENGTH: 675 <212> TYPE: DNA <213>ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS <222>LOCATION: (1)..(675) <223> OTHER INFORMATION: M2-18H <400> SEQUENCE: 97cag gtg cag ctg gtg cag tct ggg gga ggc gtg gtc cag cct ggg aag 48 GlnVal Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Lys 1 5 10 15tcc ctg aga ctc tcc tgt gca gcg tct gga ttc agc ttc agt tac tat 96 SerLeu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Tyr Tyr 20 25 30 ggcatg cac tgg gtc cgc cag gtt cca ggc aag ggg ctg gag tgg gtg 144 Gly MetHis Trp Val Arg Gln Val Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 gca gctgtc tgg tat gat gga agt act aca tat tct cca gac tcc gtg 192 Ala Ala ValTrp Tyr Asp Gly Ser Thr Thr Tyr Ser Pro Asp Ser Val 50 55 60 aag ggc cgattc acc atc tcc aga gac gat tcc aag aac acg ctg tat 240 Lys Gly Arg PheThr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr 65 70 75 80 ctg caa atgaac agc ctg aga gcc gag gac acg gct gtg tat tac tgt 288 Leu Gln Met AsnSer Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gcg aga gat agggtg ggc ctc ttt gac tac tgg ggc cag gga acc ctg 336 Ala Arg Asp Arg ValGly Leu Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 gtc acc gtc tcctca gcc tcc acc aag ggc cca tcg gtc ttc ccc ctg 384 Val Thr Val Ser SerAla Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 gca ccc tcc tccaag agc acc tct ggg ggc aca gcg gcc ctg ggc tgc 432 Ala Pro Ser Ser LysSer Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 ctg gtc aag gactac ttc ccc gaa ccg gtg acg gtg tcg tgg aac tca 480 Leu Val Lys Asp TyrPhe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 ggc gcc ctgacc agc ggc gtg cac acc ttc ccg gct gtc cta cag tcc 528 Gly Ala Leu ThrSer Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 tca gga ctctac tcc ctc agc agc gtg gtg acc gtg ccc tcc agc agc 576 Ser Gly Leu TyrSer Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 ttg ggc acccag acc tac atc tgc aac gtg aat cac aag ccc agc aac 624 Leu Gly Thr GlnThr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 acc aag gtggac aag aaa gca gag ccc aaa tct cat cac cat cac cat 672 Thr Lys Val AspLys Lys Ala Glu Pro Lys Ser His His His His His 210 215 220 cac 675 His225 <210> SEQ ID NO 98 <211> LENGTH: 225 <212> TYPE: PRT <213> ORGANISM:Homo sapiens <223> OTHER INFORMATION: M2-18H <400> SEQUENCE: 98 Gln ValGln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Lys 1 5 10 15 SerLeu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Tyr Tyr 20 25 30 GlyMet His Trp Val Arg Gln Val Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 AlaAla Val Trp Tyr Asp Gly Ser Thr Thr Tyr Ser Pro Asp Ser Val 50 55 60 LysGly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr 65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Arg Val Gly Leu Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp AsnSer 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala ValLeu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr ValPro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val AsnHis Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Lys Ala Glu Pro LysSer His His His His His 210 215 220 His 225 <210> SEQ ID NO 99 <211>LENGTH: 675 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (1)..(675) <223> OTHER INFORMATION:M2-20H <400> SEQUENCE: 99 cag gtg cag ctg gtg cag tct ggg gga ggc gtggtc cag cct ggg agg 48 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val ValGln Pro Gly Arg 1 5 10 15 tcc ctg agg ctc tcc tgt gca gcc tct gga ttcact ttc agt tac tat 96 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe ThrPhe Ser Tyr Tyr 20 25 30 ggt atg cac tgg gtc cgc cag gct cca ggc aag gggctg gag tgg gtg 144 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly LeuGlu Trp Val 35 40 45 tca ctt ata aca tat gat gga agg aat aaa tac tac gccgac tcc gtg 192 Ser Leu Ile Thr Tyr Asp Gly Arg Asn Lys Tyr Tyr Ala AspSer Val 50 55 60 aag ggc cga ttc acc atc tcc aga gag aat tcc aag aac acgctg tat 240 Lys Gly Arg Phe Thr Ile Ser Arg Glu Asn Ser Lys Asn Thr LeuTyr 65 70 75 80 ctg caa atg aac agc ctg aga act gag gac acg gct gag tattac tgt 288 Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Glu Tyr TyrCys 85 90 95 gcg aga gac ggg atc gga tac ttt gac tac tgg ggc cag gga atcctg 336 Ala Arg Asp Gly Ile Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Ile Leu100 105 110 gtc acc gtc tcc tca gcc tcc acc aag ggc cca tcg gtc ttc cccctg 384 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu115 120 125 gca ccc tcc tcc aag agc acc tct ggg ggc aca gcg gcc ctg ggctgc 432 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys130 135 140 ctg gtg aag gac tac ttc ccc gaa ccg gtg acg gtg tcg tgg aagtca 480 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Lys Ser145 150 155 160 ggc gcc ctg acc agc ggc gtg cac acc ttc ccg gct gtc ctacag tcc 528 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu GlnSer 165 170 175 tca gga ctc tac tcc ctc agc agc gtg gtg acc gtg ccc tccagc agc 576 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser SerSer 180 185 190 ttg ggc acc cag acc tac atc tgc aac gtg aat cac aag cccagc aac 624 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro SerAsn 195 200 205 acc aag gtg gac aag aaa gca gag ccc aaa tct cat cac catcac cat 672 Thr Lys Val Asp Lys Lys Ala Glu Pro Lys Ser His His His HisHis 210 215 220 cac 675 His 225 <210> SEQ ID NO 100 <211> LENGTH: 225<212> TYPE: PRT <213> ORGANISM: Homo sapiens <223> OTHER INFORMATION:M2-20H <400> SEQUENCE: 100 Gln Val Gln Leu Val Gln Ser Gly Gly Gly ValVal Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser GlyPhe Thr Phe Ser Tyr Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro GlyLys Gly Leu Glu Trp Val 35 40 45 Ser Leu Ile Thr Tyr Asp Gly Arg Asn LysTyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Glu AsnSer Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Thr GluAsp Thr Ala Glu Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Ile Gly Tyr Phe AspTyr Trp Gly Gln Gly Ile Leu 100 105 110 Val Thr Val Ser Ser Ala Ser ThrLys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser ThrSer Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr PhePro Glu Pro Val Thr Val Ser Trp Lys Ser 145 150 155 160 Gly Ala Leu ThrSer Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly LeuTyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu GlyThr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 ThrLys Val Asp Lys Lys Ala Glu Pro Lys Ser His His His His His 210 215 220His 225 <210> SEQ ID NO 101 <211> LENGTH: 675 <212> TYPE: DNA <213>ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS <222>LOCATION: (1)..(675) <223> OTHER INFORMATION: M2-31H <400> SEQUENCE: 101cag gtg cag ctg gtg gag tct ggg gga gtc gtg gtc cag cct ggg agg 48 GlnVal Gln Leu Val Glu Ser Gly Gly Val Val Val Gln Pro Gly Arg 1 5 10 15tcc ctg aga ctc tcc tgt gca gcc tct gga ttc acg ttc agt tac tat 96 SerLeu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Tyr 20 25 30 ggtata cac tgg gtc cgc cag gtt cca ggc aag gga cta gag tgg gtg 144 Gly IleHis Trp Val Arg Gln Val Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 gca cttata tca tac gat gga agc aat aaa tac tac gca gac tcc gtg 192 Ala Leu IleSer Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 aag ggc cgattc acc atc tcc aga gac aat tcc aag aac act ctg tat 240 Lys Gly Arg PheThr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 ctg caa atgaac agc ctg aga gct gag gac acg gct gtg tat tac tgt 288 Leu Gln Met AsnSer Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gcg aga gac tggatc ggg tac ttt gac tac tgg ggc cag gga acc ctg 336 Ala Arg Asp Trp IleGly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 gtc acc gtc tcctca gcc tcc acc aag ggc cca tcg gtc ttc ccc ctg 384 Val Thr Val Ser SerAla Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 gca ccc tcc tccaag agc acc tct ggg ggc aca gcg gcc ctg ggc tgc 432 Ala Pro Ser Ser LysSer Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 ctg gtc aag gactac ttc ccc gaa ccg gtg acg gtg tcg tgg aac tca 480 Leu Val Lys Asp TyrPhe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 ggc gcc ctgacc agc ggc gtg cac acc ttc ccg gct gtc cta cag tcc 528 Gly Ala Leu ThrSer Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 tca gga ctctac tcc ctc agc agc gtg gtg acc gtg ccc tcc agc agc 576 Ser Gly Leu TyrSer Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 ctg ggc acccag acc tac atc tgc aac gtg aat cac aag ccc agc aac 624 Leu Gly Thr GlnThr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 acc aag gtggac aag aaa gca gag ccc aaa tct cat cac cat cac cat 672 Thr Lys Val AspLys Lys Ala Glu Pro Lys Ser His His His His His 210 215 220 cac 675 His225 <210> SEQ ID NO 102 <211> LENGTH: 225 <212> TYPE: PRT <213>ORGANISM: Homo sapiens <223> OTHER INFORMATION: M2-31H <400> SEQUENCE:102 Gln Val Gln Leu Val Glu Ser Gly Gly Val Val Val Gln Pro Gly Arg 1 510 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Tyr 2025 30 Gly Ile His Trp Val Arg Gln Val Pro Gly Lys Gly Leu Glu Trp Val 3540 45 Ala Leu Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 5055 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 6570 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys85 90 95 Ala Arg Asp Trp Ile Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe ProLeu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala LeuGly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val SerTrp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe ProAla Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val ValThr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys AsnVal Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Lys Ala GluPro Lys Ser His His His His His 210 215 220 His 225 <210> SEQ ID NO 103<211> LENGTH: 708 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(708) <223> OTHERINFORMATION: M2-32H <400> SEQUENCE: 103 cag gtg cag ctg gtg cag tct ggggga ggc ttg gta cat cct ggg ggg 48 Gln Val Gln Leu Val Gln Ser Gly GlyGly Leu Val His Pro Gly Gly 1 5 10 15 tcc ctg aga ctc tcc tgt gaa ggctct gga ttc atc ttc agg aac cat 96 Ser Leu Arg Leu Ser Cys Glu Gly SerGly Phe Ile Phe Arg Asn His 20 25 30 cct ata cac tgg gtt cgc cag gct ccagga aaa ggt ctg gag tgg gta 144 Pro Ile His Trp Val Arg Gln Ala Pro GlyLys Gly Leu Glu Trp Val 35 40 45 tca gtt agt ggt att ggt ggt gac aca tactat gca gac tcc gtg aag 192 Ser Val Ser Gly Ile Gly Gly Asp Thr Tyr TyrAla Asp Ser Val Lys 50 55 60 ggc cga ttc tcc atc tcc aga gac aat gcc aagaac tcc ttg tat ctt 240 Gly Arg Phe Ser Ile Ser Arg Asp Asn Ala Lys AsnSer Leu Tyr Leu 65 70 75 80 caa atg aac agc ctg aga gcc gag gac atg gctgtg tat tac tgt gca 288 Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala ValTyr Tyr Cys Ala 85 90 95 aga gaa tat tac tat ggt tcg ggg agt tat cgc gttgac tac tac tac 336 Arg Glu Tyr Tyr Tyr Gly Ser Gly Ser Tyr Arg Val AspTyr Tyr Tyr 100 105 110 tac ggt atg gac gtc tgg ggc caa ggg acc acg gtcacc gtc tcc tca 384 Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val ThrVal Ser Ser 115 120 125 gcc tcc acc aag ggc cca tcg gtc ttc ccc ctg gcaccc tcc tcc aag 432 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala ProSer Ser Lys 130 135 140 agc acc tct ggg ggc aca gcg gcc ctg ggc tgc ctggtc aag gac tac 480 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu ValLys Asp Tyr 145 150 155 160 ttc ccc gaa ccg gtg acg gtg tcg tgg aac tcaggc gcc ctg acc agc 528 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser GlyAla Leu Thr Ser 165 170 175 ggc gtg cac acc ttc ccg gct gtc cta cag tcctca gga ctc tac tcc 576 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser SerGly Leu Tyr Ser 180 185 190 ctc agc agc gtg gtg acc gtg ccc tcc agc agcttg ggc acc cag acc 624 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser LeuGly Thr Gln Thr 195 200 205 tac atc tgc aac gtg aat cac aag ccc agc aacacc aag gtg gac aag 672 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn ThrLys Val Asp Lys 210 215 220 aaa gca gag ccc aaa tct cat cac cat cac catcac 708 Lys Ala Glu Pro Lys Ser His His His His His His 225 230 235<210> SEQ ID NO 104 <211> LENGTH: 236 <212> TYPE: PRT <213> ORGANISM:Homo sapiens <223> OTHER INFORMATION: M2-32H <400> SEQUENCE: 104 Gln ValGln Leu Val Gln Ser Gly Gly Gly Leu Val His Pro Gly Gly 1 5 10 15 SerLeu Arg Leu Ser Cys Glu Gly Ser Gly Phe Ile Phe Arg Asn His 20 25 30 ProIle His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 SerVal Ser Gly Ile Gly Gly Asp Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 GlyArg Phe Ser Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala Val Tyr Tyr Cys Ala 85 90 95Arg Glu Tyr Tyr Tyr Gly Ser Gly Ser Tyr Arg Val Asp Tyr Tyr Tyr 100 105110 Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115120 125 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys130 135 140 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys AspTyr 145 150 155 160 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly AlaLeu Thr Ser 165 170 175 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser SerGly Leu Tyr Ser 180 185 190 Leu Ser Ser Val Val Thr Val Pro Ser Ser SerLeu Gly Thr Gln Thr 195 200 205 Tyr Ile Cys Asn Val Asn His Lys Pro SerAsn Thr Lys Val Asp Lys 210 215 220 Lys Ala Glu Pro Lys Ser His His HisHis His His 225 230 235 <210> SEQ ID NO 105 <211> LENGTH: 675 <212>TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY:CDS <222> LOCATION: (1)..(675) <223> OTHER INFORMATION: M2-33H <400>SEQUENCE: 105 cag gtg cag ctg gtg cag tct ggg gga ggc gtg gtc cag cctggg agg 48 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro GlyArg 1 5 10 15 tcc ctg aga ctc tcc tgt gca gcg tct gga ttt acc ttc agttac tat 96 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser TyrTyr 20 25 30 ggc atg cac tgg gtc cgc cag gct cca ggc aag ggg ctg gaa tggatg 144 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met35 40 45 aca ctt ata acc tat gat gga gat aat aaa tac tat gca gac tcc gtg192 Thr Leu Ile Thr Tyr Asp Gly Asp Asn Lys Tyr Tyr Ala Asp Ser Val 5055 60 aag ggc cga ttc acc atc tcc aga gac aat tcc aag aac acg ctg tat240 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 6570 75 80 ctg caa atg aac agt ctg aga gcc gag gac acg gct gtg tat tac tgt288 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 8590 95 gcg aga gac ggg atc ggg tac ttt gac tat tgg ggc cag gga acc ctg336 Ala Arg Asp Gly Ile Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100105 110 gtc acc gtc tcc tca gcc tcc acc aag ggc cca tcg gtc ttc ccc ctg384 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115120 125 gca ccc tcc tcc aag agc acc tct ggg ggc aca gcg gcc ctg ggc tgc432 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130135 140 ctg gtc aag gac tac ttc ccc gaa ccg gtg acg gtg tcg tgg aac tca480 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145150 155 160 ggc gcc ctg acc agc ggc gtg cac acc ttc ccg gct gtc cta cagtcc 528 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser165 170 175 tca gga ctc tac tcc ctc agc agc gtg gtg acc gtg ccc tcc agcagc 576 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser180 185 190 ttg ggc acc cag acc tac atc tgc aac gtg aat cac aag ccc agcaac 624 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn195 200 205 acc aag gtg gac aag aaa gca gag ccc aaa tct cat cac cat caccat 672 Thr Lys Val Asp Lys Lys Ala Glu Pro Lys Ser His His His His His210 215 220 cac 675 His 225 <210> SEQ ID NO 106 <211> LENGTH: 225 <212>TYPE: PRT <213> ORGANISM: Homo sapiens <223> OTHER INFORMATION: M2-33H<400> SEQUENCE: 106 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val GlnPro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe ThrPhe Ser Tyr Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys GlyLeu Glu Trp Met 35 40 45 Thr Leu Ile Thr Tyr Asp Gly Asp Asn Lys Tyr TyrAla Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser LysAsn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp ThrAla Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Ile Gly Tyr Phe Asp Tyr TrpGly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys GlyPro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser GlyGly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro GluPro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser GlyVal His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr SerLeu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr GlnThr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys ValAsp Lys Lys Ala Glu Pro Lys Ser His His His His His 210 215 220 His 225<210> SEQ ID NO 107 <211> LENGTH: 675 <212> TYPE: DNA <213> ORGANISM:Homo sapiens <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION:(1)..(675) <223> OTHER INFORMATION: M2-34H <400> SEQUENCE: 107 cag gtgcag ctg gtg gag tct ggg gga ggc gtg gtc cag cct ggg agg 48 Gln Val GlnLeu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 tcc ctgaga ctc tcc tgt gca gcc tct gga ttc acg ttc agt tac tat 96 Ser Leu ArgLeu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Tyr 20 25 30 ggt ata cactgg gtc cgc cag gtt cca ggc aag gga cta gag tgg gtg 144 Gly Ile His TrpVal Arg Gln Val Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 gta ctt ata tcatac gat gga agc aat aaa tac tac gca gac tcc gtg 192 Val Leu Ile Ser TyrAsp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 aag ggc cga ttc accatc tcc aga gac aat tcc aag aac act ctg tat 240 Lys Gly Arg Phe Thr IleSer Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 ctg caa atg aac agcctg aga gct gag gac acg gct gtg tat tac tgt 288 Leu Gln Met Asn Ser LeuArg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gcg aga gac tgg atc gggtac ttt gac tac tgg ggc cag gga acc ctg 336 Ala Arg Asp Trp Ile Gly TyrPhe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 gtc acc gtc tcc tca gcctcc acc aag ggc cca tcg gtc ttc ccc ctg 384 Val Thr Val Ser Ser Ala SerThr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 gca ccc tcc tcc aag agcacc tct ggg ggc aca gcg gcc ctg ggc tgc 432 Ala Pro Ser Ser Lys Ser ThrSer Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 ctg gtc aag gac tac ttcccc gaa ccg gtg acg gtg tcg tgg aac tca 480 Leu Val Lys Asp Tyr Phe ProGlu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 ggc gcc ctg acc agcggc gtg cac acc ttc ccg gct gtc cta cag tcc 528 Gly Ala Leu Thr Ser GlyVal His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 tca gga ctc tac tccctc agc agc gtg gtg acc gtg ccc tcc agc agc 576 Ser Gly Leu Tyr Ser LeuSer Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 ctg ggc acc cag acctac atc tgc aac gtg aat cac aag ccc agc aac 624 Leu Gly Thr Gln Thr TyrIle Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 acc aag gtg gac aagaaa gca gag ccc aaa tct cat cac cat cac cat 672 Thr Lys Val Asp Lys LysAla Glu Pro Lys Ser His His His His His 210 215 220 cac 675 His 225<210> SEQ ID NO 108 <211> LENGTH: 225 <212> TYPE: PRT <213> ORGANISM:Homo sapiens <223> OTHER INFORMATION: M2-34H <400> SEQUENCE: 108 Gln ValGln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 SerLeu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Tyr 20 25 30 GlyIle His Trp Val Arg Gln Val Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 ValLeu Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 LysGly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Trp Ile Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp AsnSer 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala ValLeu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr ValPro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val AsnHis Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Lys Ala Glu Pro LysSer His His His His His 210 215 220 His 225 <210> SEQ ID NO 109 <211>LENGTH: 675 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (1)..(675) <223> OTHER INFORMATION:M2-35H <400> SEQUENCE: 109 cag gtg cag ctg gtg gag tct ggg gga ggc gtggtc cag cct ggg agg 48 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val ValGln Pro Gly Arg 1 5 10 15 tcc ctg aga ctc tcc tgt gca gcc tct gga ttcacg atc agt tac tat 96 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe ThrIle Ser Tyr Tyr 20 25 30 ggt ata cac tgg gtc cgc cag gtt cca ggc aag ggacta gag tgg gtg 144 Gly Ile His Trp Val Arg Gln Val Pro Gly Lys Gly LeuGlu Trp Val 35 40 45 gaa ctt ata tca tac gat gga agc aat aaa tac tac gcagac tcc gtg 192 Glu Leu Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala AspSer Val 50 55 60 aag ggc cga ttc acc atc tcc aga gac aat tcc aag aac actctg tat 240 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr LeuTyr 65 70 75 80 ctg caa atg aac agc ctg aga gct gag gac acg gct gtg tattac tgt 288 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr TyrCys 85 90 95 gcg aga gac tgg atc ggg tac ttt gac tac tgg ggc cag gga accctg 336 Ala Arg Asp Trp Ile Gly Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu100 105 110 gtc acc gtc tcc tca gcc tcc acc aag ggc cca tcg gtc ttc cccctg 384 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu115 120 125 gca ccc tcc tcc aag agc acc tct ggg ggc aca gcg gcc ctg ggctgc 432 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys130 135 140 ctg gtc aag gac tac ttc ccc gaa ccg gtg acg gtg tcg tgg aactca 480 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser145 150 155 160 ggc gcc ctg acc agc ggc gtg cac acc ttc ccg gct gtc ctacag tcc 528 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu GlnSer 165 170 175 tca gga ctc tac tcc ctc agc agc gtg gtg acc gtg ccc tccagc agc 576 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser SerSer 180 185 190 ctg ggc acc cag acc tac atc tgc aac gtg aat cac aag cccagc aac 624 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro SerAsn 195 200 205 acc aag gtg gac aag aaa gca gag ccc aaa tct cat cac catcac cat 672 Thr Lys Val Asp Lys Lys Ala Glu Pro Lys Ser His His His HisHis 210 215 220 cac 675 His 225 <210> SEQ ID NO 110 <211> LENGTH: 225<212> TYPE: PRT <213> ORGANISM: Homo sapiens <223> OTHER INFORMATION:M2-35H <400> SEQUENCE: 110 Gln Val Gln Leu Val Glu Ser Gly Gly Gly ValVal Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser GlyPhe Thr Ile Ser Tyr Tyr 20 25 30 Gly Ile His Trp Val Arg Gln Val Pro GlyLys Gly Leu Glu Trp Val 35 40 45 Glu Leu Ile Ser Tyr Asp Gly Ser Asn LysTyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp AsnSer Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala GluAsp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Trp Ile Gly Tyr Phe AspTyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser ThrLys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser ThrSer Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr PhePro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu ThrSer Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly LeuTyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu GlyThr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 ThrLys Val Asp Lys Lys Ala Glu Pro Lys Ser His His His His His 210 215 220His 225

What is claimed is:
 1. A method of detecting an analyte in a humansample containing human antibodies that specifically bind to antibodiesfrom a nonhuman species, comprising: contacting the sample with a humanantibody, wherein the human antibody specifically binds to the analytewithout specifically binding to the human antibodies that specificallybind to antibodies from a nonhuman species, and detecting the bindingbetween the human antibody and the analyte to indicate presence of theanalyte.
 2. The method of claim 1, wherein the human sample containshuman anti-mouse antibodies.
 3. The method of claim 2, wherein the humananti-mouse antibodies are anti-mouse idiotype antibodies.
 4. The methodof claim 1, wherein the human sample contains heterophilic antibodies.5. The method of claim 1, wherein the sample is contacted with a firsthuman antibody that is immobilized on a support and a second humanantibody in solution wherein the first and second human antibodies bindto different epitopes on the analyte; and the detecting step detectsbinding between the first and second human antibody to the analyte. 6.The method of claim 5, wherein the detecting step detects bindingbetween the second human antibody and the analyte.
 7. The method ofclaim 6, wherein the second human antibody is labelled.
 8. The method ofclaim 6, wherein the sample is contacted with a first population ofhuman antibodies immobilized to a support and a second population ofhuman antibodies in solution, wherein members from the first and secondpopulations bind to different epitopes on the analyte.
 9. The method ofclaim 8, wherein the second population of human antibodies is labelled.10. The method of claim 5, wherein the first and second human antibodieseach have affinities of at least 10⁹ M⁻¹ for their respective epitopeson the analyte.
 11. The method of claim 5, wherein the first and secondhuman antibodies are produced by subcloning nucleic acids encoding thefirst and second human antibodies from a transgenic mouse expressinghuman immunoglobulin genes into a phage display vector, and screeningphage displaying the first human antibodies and phage displaying thesecond human antibodies for desired binding specificities.
 12. Themethod of claim 1, wherein the human antibody has an affinity of atleast 10⁸ M⁻¹ for the analyte.
 13. The method of claim 1, wherein thehuman antibody has an affinity of at least 10⁹ M⁻¹ for the analyte. 14.The method of claim 1, wherein the human antibody has an affinity of atleast 10¹⁰ M⁻¹ for the analyte.
 15. The method of claim 1, wherein thehuman antibody has an affinity of a least 10¹¹M⁻¹ for the analyte. 16.The method of claim 1, wherein binding of the human antibody to theanalyte reaches equilibrium within an hour.
 17. The method of claim 1,wherein the antibody was produced by expression by expression of arecombinant construct in E. coli.
 18. The method of claim 17, wherein atleast 90% of molecules of the antibody are immunoreactive with theanalyte.
 19. A method of detecting an analyte in a sample, comprisingcontacting the sample with a first human antibody immobilized to a solidphase, and a second human antibody in solution, wherein the first andsecond antibodies bind to different epitopes of the analyte if presentin the sample detecting binding of the analyte to the first and secondantibodies, binding indicating presence of the analyte in the samplewherein the first and second human antibodies are produced by subcloningnucleic acids encoding the first and second human antibodies from atransgenic mouse expressing human immunoglobulin genes into a phagedisplay vector, and screening phage displaying the first humanantibodies and phage displaying the second human antibodies for desiredbinding specificities.
 20. The method of claim 19, wherein the secondantibody is labelled and the detecting detects binding of secondantibody to the analyte.
 21. The method of claim 20, wherein the sampleis contacted with a first population of human antibodies immobilized tothe solid phase and a second population of human antibodies in solution,wherein members from the first and second populations bind to differentepitopes on the analyte.
 22. The method of claim 21, wherein the secondpopulation of human antibodies is labelled.
 23. The method of claim 19,wherein the first and second human antibodies each have an affinity ofat least 10⁹ M⁻¹ for their respective epitopes on the analyte.
 24. Themethod of claim 19, wherein the first and second human antibodies eachhave an affinity of at least 10¹⁰ M⁻¹ for the analyte.
 25. The method ofclaim 19, wherein the first and second human antibodies have an affinityof a least 10¹¹M⁻¹ for the analyte.
 26. The method of claim 19, whereinbinding of the first and second human antibodies to the analyte reachesequilibrium within an hour.
 27. The method of claim 19, wherein thefirst and second human antibodies were produced by expression byexpression of recombinant constructs in E. coli.
 28. The method of claim27, wherein at least 90% of molecules of the first and second antibodyare immunoreactive with the analyte.